MedWorm.com provides a medical RSS filtering service. Over 6000 RSS medical sources are combined and output via different filters. This feed contains the latest items from the 'Springer protocols feed by Cell Biology' source. Tue, 07 Feb 2012 08:49:02 +0100 http://www.medworm.com/index.php?rid=5618304&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-527-5_20 Nitric oxide (NO) is enzymatically produced from l-arginine and has a variety of biological functions. Autoxidation of NO in aqueous media yields nitrite (O = N–O−). NO and nitrite are oxidized in erythrocytes by oxyhemoglobin to nitrate (NO 3 − ). Nitrate reductases from bacteria reduce nitrate to nitrite. Nitrite and nitrate are ubiquitous in nature, they are present throughout the body and they are excreted in the urine. Nitrite in urine has been used for several decades as an indicator and measure of bacteriuria. Since the identification of nitrite as a metabolite of NO, circulating nitrite is also used as an indicator of NO synthesis and is considered an NO storage form. In contrast to plasma nitrite, the significance of nitrite in the urine be... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5618304 Sun, 01 Jan 2012 05:00:00 +0100 5618304 http://www.medworm.com/index.php?rid=5618303&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-527-5_1 Deciphering the contribution of individual genes and in turn pathways to cellular processes can be ­complicated and is often based on prior knowledge or assumptions of gene function. Phenotype-driven mutagenesis screens based around n-ethyl-n-nitrosurea (ENU) have been successful in a wide range of physiological systems in identifying novel genes that contribute to a given phenotype. Here, we describe methodologies we have employed in analysing cellular phenotypes in pipelines of mutagenised mice. Examples of primary screens to identify outliers, and secondary screens to provide a more detailed characterisation are outlined. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5618303 Sun, 01 Jan 2012 05:00:00 +0100 5618303 http://www.medworm.com/index.php?rid=5618302&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-527-5_19 Inflammatory bowel diseases (IBD) consist of Crohn’s disease (CD) and ulcerative colitis (UC) affecting about 0.1% of the western population. These two chronic gut diseases affect youth at their prime of life causing diarrhoea, intestinal bleeding, and severe gut discomfort. Mouse models of colitis have been major tools in understanding the pathogenesis of IBD. A number of mouse models are available to assess the contribution of T cells in the pathogenesis of CD and UC. Among these, the T cell transfer model of colitis is the most widely used model to dissect the initiation, induction, and regulation of immunopathology in chronic colitis mediated by T cells. The methodology below describes the classification of various animal models and explains the T cell transfer model in detail, i... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5618302 Sun, 01 Jan 2012 05:00:00 +0100 5618302 http://www.medworm.com/index.php?rid=5618301&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-527-5_18 The serine protease granzyme B (GrB) is a key effector molecule in cell-mediated immunity, released by cytotoxic lymphocytes (CLs) to induce cell death in neoplastic or virus-infected cells. The ability to detect and measure GrB activity is important for understanding CLs. Unfortunately, such analyses are complicated by significant differences in the substrate specificities of human and mouse GrB, which is reflected by their different activities on commonly used peptide substrates. Here, we present methods for the detection of active human and mouse GrB in extracts from primary cells, and evaluate the sensitivity of the various substrates and inhibitors. Mouse splenocytes produce approximately 120-fold more GrB than similarly activated human cells, which allows the use of the hGrB substrat... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5618301 Sun, 01 Jan 2012 05:00:00 +0100 5618301 http://www.medworm.com/index.php?rid=5618300&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-527-5_17 Investigation of Granzyme B (GrB) function and pathophysiology in both human settings and rodent models increasingly involve the use of indirect immunofluorescence imaging and fluorescence-activated cell sorting, which requires reliable GrB antibodies that do not recognise other closely related granzymes. Here, we describe the validation (using a set of recombinant granzymes, and GrB-deficient cells) and application of widely available monoclonal antibodies to specifically monitor GrB in human or mouse cells. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5618300 Sun, 01 Jan 2012 05:00:00 +0100 5618300 http://www.medworm.com/index.php?rid=5618299&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-527-5_2 Accurate measurement of cytokine concentrations is a powerful and essential approach to the study of inflammation. The enzyme-linked immunosorbent assay (ELISA) is a simple, low-cost analytical tool that provides both the specificity and sensitivity required for the study of cytokines in vitro or in vivo. This communication describes a systematic approach to develop an indirect sandwich ELISA to detect and quantify cytokines, or other biomarkers, with accuracy and precision. Also detailed is the use of sequential ELISA assays to analyze multiple cytokines from samples with limited volumes. Finally, the concept of a multiplex ELISA is discussed with considerations given to cost and additional time required for development. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5618299 Sun, 01 Jan 2012 05:00:00 +0100 5618299 http://www.medworm.com/index.php?rid=5618298&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-527-5_5 Cell death is of utmost importance in immunity, in part as a way to control the development and activity of leukocytes, but also as a strategy employed by leukocytes to rid the body of unwanted cells. Apoptosis is the classic type of programmed cell death involving an ordered sequence of cellular events, resulting in morphological changes that include cleavage/fragmentation of DNA, condensation of nuclei, cell shrinkage, and alterations of the plasma membrane. The apoptotic cell is a nonfunctional, but structurally intact, entity with preserved membrane integrity that is engulfed by surrounding cells (a process known as clearance) in an immunologically silent manner. In contrast, necrotic cells, i.e., nonfunctional cells that have lost membrane integrity, are freely permeable and leak intr... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5618298 Sun, 01 Jan 2012 05:00:00 +0100 5618298 http://www.medworm.com/index.php?rid=5618297&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-527-5_4 Identification of the precise location, where hematopoietic stem cells (HSCs) reside in the bone marrow, has made a great leap forward with the advance of live time-lapse video 2-photon fluorescent microscopy. These studies have shown that HSCs preferentially resides in the endosteal region of the BM, at an average of two cell diameters from osteoblasts covering endosteal bone surfaces. However, this equipment is very sophisticated and only a very few laboratories can perform these studies. To investigate functional attributes of these niches, we have developed a flow cytometry technique in which mice are perfused with the cell-permeable fluorescent dye Hoechst33342 in vivo before bone marrow cells are collected and antibody stained. This method enables to position phenotypic HSC, multipot... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5618297 Sun, 01 Jan 2012 05:00:00 +0100 5618297 http://www.medworm.com/index.php?rid=5618296&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-527-5_3 The hematopoietic system is highly proliferative in the bone marrow (BM) due to the short half-life of granulocytes and platelets in the blood. Analysis of cell cycling and cell proliferation in vivo in specific populations of the mouse BM has highlighted some key properties of adult hematopoietic stem cells (HSCs). For instance, despite their enormous proliferation and repopulation potential, most true HSC are deeply quiescent in G0 phase of the cell cycle and divide very infrequently, while less potent lineage-restricted progenitors divide rapidly to replace the daily consumption of blood leukocytes, erythrocytes, and platelets. In response to stress, e.g., following ablative chemotherapy or irradiation, HSC must enter the cell cycle to rapidly repopulate the BM with progenitors. Due to ... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5618296 Sun, 01 Jan 2012 05:00:00 +0100 5618296 http://www.medworm.com/index.php?rid=5466845&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-430-8_5 The mammalian Target Of Rapamycin (mTOR) protein is a central component of the essential and highly conserved signaling pathway that emerged as a critical effector in regulation of cell physiology. Biochemical studies defined mTOR as the protein kinase that exists at least in two distinct complexes. The first complex has been characterized as the nutrient-sensitive mTOR complex 1 that controls cell growth and cell size by regulating protein synthesis and autophagy. The second complex of mTOR has been defined as the component of growth factor signaling that functions as a major regulatory kinase of Akt/PKB. Here, we provide the detailed methods how to purify the functional complexes of mTOR by affinity purification. In the first part, we describe the purification of the distinct mTOR comple... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5466845 Sat, 03 Dec 2011 02:54:59 +0100 5466845 http://www.medworm.com/index.php?rid=5466844&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-430-8_4 The adaptive response to hypoxia, low oxygen tension, involves inhibition of energy-intensive cellular processes including protein translation. This effect is mediated in part through a decrease in the kinase activity of mammalian target of rapamycin complex 1 (mTORC1), a master regulator of protein translation. The principle mechanism for hypoxia-induced mTORC1 inhibition, however, was not elucidated until recently. Our work has demonstrated that the stress-induced protein REDD1 is essential for hypoxia regulation of mTORC1 activity and has further defined the molecular mechanism whereby REDD1 represses mTORC1 activity under hypoxic stress. Using our studies with REDD1 as an example, we describe in detail biochemical approaches to assess mTORC1 activity in the hypoxic response. Here, we p... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5466844 Sat, 03 Dec 2011 02:54:59 +0100 5466844 http://www.medworm.com/index.php?rid=5466843&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-430-8_3 mTOR, an evolutionarily conserved Ser/Thr protein kinase, belongs to the PI3K-related kinase family, which also includes DNA-PKcs, ATM, and ATR. Although other PI3K-related kinase family members have been shown to secure genomic integrity by sensing DNA damage and related stresses, mTOR is known to function as a nutrient and growth factor sensor. mTOR is the catalytic subunit of two distinct multiprotein complexes known as mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). In response to growth factor and nutrient availability, these complexes regulate a variety of cellular processes, such as cell growth, proliferation, and survival by modulating downstream effectors, such as S6K1, 4EBP1, and AKT. Therefore, evaluation of mTOR activity has been a clear readout in order to monitor the phy... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5466843 Sat, 03 Dec 2011 02:54:59 +0100 5466843 http://www.medworm.com/index.php?rid=5466842&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-430-8_2 The mammalian target of rapamycin (mTOR) is the catalytic subunit of two multiprotein complexes, mTOR complex-1 (mTORC1) and mTOR complex-2 (mTORC2). Clinically used rapamycin and rapalogs are FKBP12-dependent allosteric inhibitors of mTORC1. The recently discovered WYE-125132 and related drugs represent a new generation of ATP competitive and highly specific inhibitors targeting mTOR globally. As mTORC1 and mTORC2 mediate diverse sets of both redundant and distinctive cellular pathways of growth, nutrient and energy homeostasis, rapamycin and WYE-125132 elicit both overlapping and distinctive pharmacological properties with important implications in treating cancer, metabolic, and age-related degenerative diseases. Detailed methods are described for the determination of mTOR inhibition by... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5466842 Sat, 03 Dec 2011 02:54:59 +0100 5466842 http://www.medworm.com/index.php?rid=5466841&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-430-8_29 The mammalian Target of Rapamycin (mTOR)-mediated signaling transduction pathway has been observed to be deregulated in a wide variety of cancer and metabolic diseases. Despite extensive clinical development efforts, the well-known allosteric mTOR inhibitor rapamycin and structurally related rapalogs have failed to show significant single-agent antitumor efficacy in most types of cancer. This limited clinical success may be due to the inability of the rapalogs to maintain a complete blockade mTOR-mediated signaling. Therefore, numerous efforts have been initiated to develop ATP-competitive mTOR inhibitors that would block both mTORC1 and mTORC2 complex activity. Here, we describe our experimental approaches to develop Torin1 using a medium throughput cell-based screening assay and structur... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5466841 Sat, 03 Dec 2011 02:54:59 +0100 5466841 http://www.medworm.com/index.php?rid=5466840&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-430-8_28 The potent immunosuppressive action of rapamycin has been described in many different mouse models of transplantation. In these models, rapamycin prevent or delay allograft rejection. In several models, rapamycin allowed mixed donor–recipient hematopoietic chimerism to develop facilitating tolerance induction. In our own experience, we observed that rapamycin synergized with CD8+ T cell depletion and coreceptor/costimulation blockade to induce long-term survival of Balb/C to C57Bl/6 heterotopic limb allograft. Herein, we describe immunosuppression cocktails containing rapamycin and methods to evaluate several read outs associated with tolerance induction such as mixed donor–recipient hematopoietic chimerism and in vitro or in vivo recipient alloreactivity. (Source: Springer pro... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5466840 Sat, 03 Dec 2011 02:54:59 +0100 5466840 http://www.medworm.com/index.php?rid=5466839&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-430-8_27 Obesity is reaching pandemic proportions in Western society. It has resulted in increasing health care burden and decreasing life expectancy. Obesity is a complex, chronic disease, involving decades of pathophysiological changes and adaptation. Therefore, it is difficult ascertain the exact mechanisms for this long-term process in humans. To circumvent some of these issues, several surrogate models are available, including murine genetic loss-of-function mutations, transgenic gain-of-function mutations, polygenic models, and different environmental exposure models. The mouse model of diet-induced obesity has become one of the most important tools for understanding the interplay of high-fat Western diets and the development of obesity. The diet-induced obesity model closely mimics the incre... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5466839 Sat, 03 Dec 2011 02:54:59 +0100 5466839 http://www.medworm.com/index.php?rid=5466838&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-430-8_26 Tuberous sclerosis complex 1 (TSC1) is a tumor suppressor that associates with TSC2 to inactivate Rheb, thereby inhibiting signaling by the mammalian target of rapamycin (mTOR) complex 1 (mTORC1). mTORC1 stimulates cell growth by promoting anabolic cellular processes, such as translation, in response to growth factors and nutrient signals. In order to test roles for TSC1 and mTORC1 in β-cell function, we utilized Rip2/Cre to generate mice lacking Tsc1 in pancreatic β cells (Rip-Tsc1cKO mice). While obesity developed due to hypothalamic Tsc1 excision in older Rip-Tsc1cKO animals, young animals displayed a prominent gain-of-function β-cell phenotype prior to the onset of obesity. The young Rip-Tsc1cKO animals displayed improved glycemic control due to mTOR-mediated enhancement... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5466838 Sat, 03 Dec 2011 02:54:59 +0100 5466838 http://www.medworm.com/index.php?rid=5466837&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-430-8_25 Tuberous sclerosis complex (TSC) is an autosomal dominant disease caused by mutations in either of two genes, TSC1 or TSC2, whose protein products form a complex that is essential in the regulation of mammalian target of rapamycin (mTOR) activity. TSC is characterized by the presence of benign tumors called hamartomas, which within the brain are known as cortical tubers. Neurological manifestations in TSC patients include epilepsy, mental retardation, and autistic features. In response to hormones, growth factors, or nutrients, the phosphatidylinositol 3-kinase or extracellular signal-regulated kinase-Tsc-mTOR pathways activate the translation machinery and regulate cell growth and/or size. Loss of TSC1 or TSC2 function results in constitutive activation of mTOR leading to tumor formation.... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5466837 Sat, 03 Dec 2011 02:54:59 +0100 5466837 http://www.medworm.com/index.php?rid=5466836&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-430-8_24 Tuberous Sclerosis Complex (TSC) is a genetic disease involving dysregulation of the mTOR pathway and resulting in disabling neurological manifestations, such as epilepsy. Animal models may recapitulate epilepsy and other behavioral features of TSC and are useful tools for investigating mechanisms of epileptogenesis and other neurological deficits in TSC. In this chapter, methods for performing video-electroencephalography (video-EEG) to characterize epilepsy and neurological dysfunction in rodent models are reviewed. In particular, technical aspects of surgical implantation of EEG electrodes, video-EEG recording, and analysis and interpretation of EEG data are detailed. These methodological approaches should be helpful in characterizing seizures and background EEG abnormalities not only i... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5466836 Sat, 03 Dec 2011 02:54:59 +0100 5466836 http://www.medworm.com/index.php?rid=5466835&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-430-8_23 We described a protocol for dissecting the function of an important serine/threonine protein kinase, mammalian target of rapamycin (mTOR), in regulating the long-term undifferentiated growth of human embryonic stem cells (hESCs). The function of mTOR in hESCs was inactivated with a highly specific chemical inhibitor, rapamycin, and gene-specific small-hairpin RNAs, and the effects were evaluated under self-renewal or early differentiation conditions. We found that inactivation of mTOR impairs proliferation and enhances mesoderm and endoderm activities of hESCs. This protocol described a general strategy for studying the function of key genes and signaling events during hESC long-term self-renewal and early lineage specifications with pharmacological and genetic approaches. (Source: Springe... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5466835 Sat, 03 Dec 2011 02:54:59 +0100 5466835 http://www.medworm.com/index.php?rid=5466834&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-430-8_22 Amplification of the gene encoding the epidermal growth factor receptor (EGFR) occurs commonly in glioblastoma (GBM), leading to activation of downstream kinases, including phosphatidylinositol 3′-kinase (PI3K), Akt, and mammalian target of rapamycin (mTOR). A serine-threonine kinase, mTOR controls cell growth by regulating mRNA translation, metabolism, and autophagy; acting as both a downstream effector and upstream regulator of PI3K. These signaling functions are distributed between at least two distinct complexes, mTORC1 and mTORC2 with respect to pathway specificity. We have investigated mTOR signaling in glioma cells with the allosteric mTORC1 inhibitor rapamycin, the mTORC1/2 inhibitor Ku-0063794, a dual PI3K/mTORC1/2 kinase inhibitor PI-103, and siRNA against raptor, rictor, o... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5466834 Sat, 03 Dec 2011 02:54:59 +0100 5466834 http://www.medworm.com/index.php?rid=5466833&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-430-8_21 Extensive efforts are underway to develop small-molecule inhibitors of the mammalian target of rapamycin (mTOR) kinase. It is hoped that these inhibitors will have widespread clinical impact in oncology because mTOR is a major downstream effector of PI3K signaling, one of the most frequently activated pathways in cancer. In cells, mTOR is the catalytic core subunit of two distinct complexes, mTORC1 and mTORC2, which are defined by unique mTOR-interacting proteins and have unique functions downstream of PI3K. Two classes of mTOR inhibitors are currently being evaluated as cancer therapeutics: rapamycin and its analogs, which partially inhibit mTORC1 and in some cell types mTORC2, and the recently described ATP-competitive inhibitors, which inhibit the kinase activity of both complexes. Alth... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5466833 Sat, 03 Dec 2011 02:54:59 +0100 5466833 http://www.medworm.com/index.php?rid=5466832&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-430-8_20 The mammalian Target of Rapamycin (mTOR) defines a crucial link between nutrient sensing and immune function. In CD4+ T cells, mTOR has been shown to play a critical role in regulating effector and regulatory T cell differentiation as well as the decision between full activation versus the induction of anergy. In this chapter, we describe how our group has employed the Cre-lox technology to genetically delete components of the mTOR signaling complex in T cells. This has enabled us to specifically interrogate mTOR function in T cells both in vitro and in vivo. We also describe techniques used to assay immune function and signaling in mTOR-deficient T cells at the single-cell level. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5466832 Sat, 03 Dec 2011 02:54:59 +0100 5466832 http://www.medworm.com/index.php?rid=5466831&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-430-8_1 The mammalian (or mechanistic) target of rapamycin (mTOR) is an evolutionarily conserved serine-threonine kinase that is known to sense the environmental and cellular nutrition and energy status. Diverse mitogens, growth factors, and nutrients stimulate the activation of the two mTOR complexes mTORC1 and mTORC2 to regulate diverse functions, such as cell growth, proliferation, development, memory, longevity, angiogenesis, autophagy, and innate as well as adaptive immune responses. Dysregulation of the mTOR pathway is frequently observed in various cancers and in genetic disorders, such as tuberous sclerosis complex or cystic kidney disease. In this review, I will give an overview of the current understanding of mTOR signaling and its role in diverse tissues and cells. Genetic deletion of s... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5466831 Sat, 03 Dec 2011 02:54:59 +0100 5466831 http://www.medworm.com/index.php?rid=5466830&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-430-8_19 RNA interference (RNAi) is an intracellular mechanism for silencing gene expression utilizing short fragments of double-strand RNA that are complementary to the target messenger RNA. This gene silencing technique has now become an invaluable research tool due to its specific and strong repressive effect on a target transcript. We have recently applied a retrovirus-based RNAi system to investigate the in vivo role of the mammalian target of rapamycin (mTOR) in antigen-specific CD8 T cells, and have found that mTOR regulates memory CD8 T-cell differentiation. Here, we provide a detailed protocol for knocking down mTOR and its related molecules (raptor and FKBP12) in antigen-specific CD8 T cells. In our protocol, a mouse model of lymphocytic choriomeningitis virus infection is used, but the m... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5466830 Sat, 03 Dec 2011 02:54:59 +0100 5466830 http://www.medworm.com/index.php?rid=5466829&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-430-8_18 Th1 immunity protects against tuberculosis infection in mice and humans. The widely used BCG vaccine primes CD4 and CD8 T cells through signaling mechanisms from dendritic cells and macrophages. The latter express MHC-II and MHC-I molecules through which peptides from BCG vaccine are presented to CD4 and CD8 T cells, respectively. Since BCG sequesters within a phagosome that does not fuse with lysosomes, generation of peptides within antigen-presenting cells infected with BCG occurs with reduced efficiency. We demonstrate that activation of DCs containing BCG vaccine with rapamycin leads to an enhanced ability of DC vaccines to immunize mice against tuberculosis. Coadministration of rapamycin with BCG vaccine also enhanced Th1 immunity. We propose that rapamycin-mediated increase in Th1 re... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5466829 Sat, 03 Dec 2011 02:54:59 +0100 5466829 http://www.medworm.com/index.php?rid=5466828&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-430-8_17 CD4+CD25+FOXP3+ T regulatory (Treg) cells are pivotal for the induction and maintenance of peripheral tolerance in both mice and humans. The possibility to use Treg cells for the treatment of T-cell-mediated diseases has recently gained increasing momentum. However, given the limited amount of circulating FOXP3+ Treg cells, efficient methods for their ex vivo expansion are highly desirable. Rapamycin allows for in vitro expansion of murine and human FOXP3+ Treg cells, which maintain their regulatory phenotype and suppressive capacity. Here, we describe in detail the powerful methods for enriching human FOXP3+ Treg cells starting from unfractionated CD4+ T cells or for expanding CD25+-enriched Treg cells in the presence of rapamycin. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5466828 Sat, 03 Dec 2011 02:54:59 +0100 5466828 http://www.medworm.com/index.php?rid=5466827&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-430-8_16 The mammalian Target of Rapamycin (mTOR) kinase functions within two structurally and functionally distinct multiprotein complexes termed mTOR complex 1 (mTORC1) and mTORC2. The immunosuppressant and anticancer drug rapamycin is commonly used in basic research as a tool to study mTOR signaling. However, rapamycin inhibits only, and only incompletely, mTORC1, and no mTORC2-specific inhibitor is available. Hence, a full understanding of mTOR signaling in vivo, including the function of both complexes, requires genetic inhibition in addition to pharmacological inhibition. Taking advantage of the Cre/LoxP system, we generated inducible knockout mouse embryonic fibroblasts (MEFs) deficient for either the mTORC1-specific component raptor (iRapKO) or the mTORC2-specific component rictor (iRicKO).... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5466827 Sat, 03 Dec 2011 02:54:59 +0100 5466827 http://www.medworm.com/index.php?rid=5466826&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-430-8_15 Preclinical evaluation of candidate anticancer compounds requires appropriate animal models. Most commonly, solid tumor xenograft systems are employed in which immunocompromised mice are implanted with human cancer cell lines. Genetically engineered mouse models of solid tumors are also frequently employed. Both of these approaches can also be applied to studies of hematological malignancies. In this chapter, we describe three types of mouse models of leukemia driven by the human BCR-ABL oncogene. We also discuss the application of these models to preclinical testing of active-site TOR inhibitors, a novel class of compounds that selectively target the ATP-binding pocket of the target of rapamycin (TOR) kinase. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5466826 Sat, 03 Dec 2011 02:54:59 +0100 5466826 http://www.medworm.com/index.php?rid=5466825&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-430-8_14 Autophagy is a catabolic pathway that degrades bulk cytosol in lysosomal compartments enabling amino acids and fatty acids to be recycled. One of the key regulators of autophagy is the mammalian target of rapamycin (mTOR), a conserved serine/threonine kinase which suppresses the initiation of the autophagic process when nutrients, growth factors, and energy are available. Inhibition of mTOR, e.g., by small molecules such as rapamycin, results in activation of autophagy. To quantify autophagy induction by mTOR inhibitors, we use an mCherry-GFP-LC3 reporter which is amenable to retroviral delivery into mammalian cells, stable expression, and analysis by fluorescence microscopy. Here, we describe our imaging protocol and image recognition algorithm to visualize and measure changes in the auto... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5466825 Sat, 03 Dec 2011 02:54:59 +0100 5466825 http://www.medworm.com/index.php?rid=5466824&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-430-8_13 Target of rapamycin (TOR) regulates the growth of cells and organisms. Numerous growth-promoting and growth-arresting pathways converge on TOR; TOR acts as an important hub, balancing the pro- and anti-growth signals within a cell. Since it regulates growth at the cellular level, cell size can be used as an indirect readout of TOR activity. Here, we describe methods used to analyze cell size in cell culture and in the Drosophila wing. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5466824 Sat, 03 Dec 2011 02:54:59 +0100 5466824 http://www.medworm.com/index.php?rid=5466823&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-430-8_12 mTOR is a key regulator of cell growth and size, and its activity is often dysregulated in a wide variety of diseases. The mTOR signaling pathway is also a therapeutic target for many diseases, including cancer. Immunohistochemistry is a powerful method to assess mTOR activity in clinical/histological samples, however, care should be taken in choosing the targets for determining mTOR activity due to the complexity of its regulation. This chapter describes the most up-to-date methods for visualizing mTOR activity by immunohistochemistry using commercially available antibodies, including considerations for validating new antibodies for assessing mTOR signaling. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5466823 Sat, 03 Dec 2011 02:54:59 +0100 5466823 http://www.medworm.com/index.php?rid=5466822&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-430-8_11 Mammalian target of rapamycin (mTOR) is a giant protein kinase that controls cell proliferation, growth, and metabolism. mTOR is regulated by nutrient availability, by mitogens, and by stress, and operates through two independently regulated hetero-oligomeric complexes. We have attempted to identify the cellular components necessary to maintain the activity of mTOR complex 1 (mTORC1), the amino acid-dependent, rapamycin-inhibitable complex, using a whole genome approach involving RNAi-induced depletion of cellular polypeptides. We have used a pancreatic ductal adenocarcinoma (PDAC) cell line, Mia-PaCa for this screen; as with many pancreatic cancers, these cells exhibit constitutive activation of mTORC1. PDAC is the most common form of pancreatic cancer and the 5-year survival rate remains... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5466822 Sat, 03 Dec 2011 02:54:59 +0100 5466822 http://www.medworm.com/index.php?rid=5466821&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-430-8_10 mTOR, the mammalian target of rapamycin, regulates protein synthesis (mRNA translation) by affecting the phosphorylation or activity of several translation factors. Here, we describe methods for studying the impact of mTOR signalling on protein synthesis, using inhibitors of mTOR such as rapamycin (which impairs some of its functions) or mTOR kinase inhibitors (which probably block all functions).To assess effects of mTOR inhibition on general protein synthesis in cells, the incorporation of radiolabelled amino acids into protein is measured. This does not yield information on the effects of mTOR on the synthesis of specific proteins. To do this, two methods are described. In one, stable-isotope labelled amino acids are used, and their incorporation into new proteins is determined using ma... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5466821 Sat, 03 Dec 2011 02:54:59 +0100 5466821 http://www.medworm.com/index.php?rid=5398364&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-367-7_7 Major hepatic cytochrome P450 activities (CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4) can be simultaneously examined in human hepatocytes by incubation with a cocktail of multiple specific probes. Cocktail strategy in combination with mass spectrometry is shown to be a robust, fast, and sensitive procedure for P450 activity assessment. This procedure allows a drastic reduction of the number of cells required in the assay and sample analysis time and increases throughput and reproducibility. Major applications of the probe cocktail strategy are P450 phenotyping of hepatocytes and induction studies. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5398364 Sat, 12 Nov 2011 02:41:18 +0100 5398364 http://www.medworm.com/index.php?rid=5398363&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-367-7_6 Type I diabetes results from the autoimmune destruction of the insulin-secreting pancreatic β-cells, affecting many millions of people worldwide. The optimal treatment is to restore the endogenous supply of insulin either through the transplantation of pancreas or the transplantation of islets of langerhans or simply the β-cells. However, the donated pancreas organs are limited and the available organs are only able to treat a small portion of the diabetes patients. Thus, glucose-responsive, insulin-producing cells from human origin are urgently needed. The aim of this chapter is to give some insight views to how to turn the potential human pancreatic non-endocrine cells into cells that are capable of secreting insulin in response to glucose and ameliorating insulin-deficient dia... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5398363 Sat, 12 Nov 2011 02:41:18 +0100 5398363 http://www.medworm.com/index.php?rid=5398362&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-367-7_5 Islets of Langerhans isolated from experimental animals, such as mice and rats, have contributed much to our understanding of the mechanisms by which pancreatic β-cells secrete insulin in a regulated manner, and this knowledge is important in identifying potential novel therapies for Type 2 diabetes. However, although many of the signal transduction pathways identified in rodent islets are common to humans, some critical differences have been demonstrated experimentally. It is, therefore, essential that experiments are performed using islets isolated from human pancreas to provide robust data defining whether the key observations made in rodents are also applicable to the human situation. The rate-limiting factor in this area of research is the supply of high-quality human islets isol... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5398362 Sat, 12 Nov 2011 02:41:18 +0100 5398362 http://www.medworm.com/index.php?rid=5398361&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-367-7_4 The parathyroid cells are highly differentiated with more or less their only function to secrete parathyroid hormone in response to the extracellular calcium level. Tumours from the parathyroid glands are >99% benign, and have a slow proliferation rate. Culture of parathyroid cells is known to be very difficult most likely due to the high differentiation level. This chapter reveals some details in order how to get parathyroid cells to survive in culture after dispersion of normal bovine parathyroid glands or pathological human parathyroid tumours. Detailed protocols describing cell dispersion with collagenase, short-term cultures, and establishment of long-term cultures are presented. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5398361 Sat, 12 Nov 2011 02:41:18 +0100 5398361 http://www.medworm.com/index.php?rid=5398360&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-367-7_3 The blood–air barrier formed by the alveolar epithelium of the peripheral lung is crucial for the pulmonary delivery of drugs. Most existing in vitro models mimicking the blood–air barrier are represented by tumor cells or immortalized cells and lack biological relevance due to their genetic alterations and underexpressed essential physiological functions. However, the increasing interest of aerosol administration of medicines to the respiratory system requires the development and use of representative in vitro models. Thereby, human alveolar epithelial cells (hAEpC) are a suitable test system allowing standardized toxicity and transport studies for newly developed compounds and delivery systems. The isolation, purification, and cultivation of hAEpC are described as well as the... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5398360 Sat, 12 Nov 2011 02:41:18 +0100 5398360 http://www.medworm.com/index.php?rid=5398359&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-367-7_2 Human melanocytes have been extensively studied, but a melanocyte stem cell reservoir in glabrous skin has not yet been found. Human dermis contains cells that are nonpigmented but can differentiate to several different cell types. We have recently shown that multipotent dermal stem cells isolated from human neonatal foreskins are able to differentiate to multiple cell lineages, including pigmented melanocytes. The dermal stem cells grow as three-dimensional spheres in human embryonic stem cell medium and express some neural crest stem cell and embryonic stem cell markers. Melanocytes derived from dermal stem cells express melanocytic markers and act the same way as mature epidermal melanocytes. Dermal spheres, embedded in the reconstructed dermis consisting of collagen with fibroblasts, c... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5398359 Sat, 12 Nov 2011 02:41:18 +0100 5398359 http://www.medworm.com/index.php?rid=5398358&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-367-7_27 Mesenchymal stem cells from a variety of sites are a natural resource that using appropriate skills can be cultured in the laboratory, in scaffolds, to provide differentiated-cell replacement tissues, for clinical application. To perform such work with human cells, strict ethical integrity must be observed at all stages. Adipocytes, osteocytes and chrondrocytes are amongst the most desirable end-point cells. Hydrolytic degradable scaffolds allow implanted cells to synthesise their own extracellular matrix in situ after implantation, degeneration of the foreign scaffold to temporally match creation of the new innate one. For preliminary in vitro stem cell differentiation protocols, initial investigation is commonly performed with stem cells in commercially available porous collagen sponges ... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5398358 Sat, 12 Nov 2011 02:41:18 +0100 5398358 http://www.medworm.com/index.php?rid=5398357&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-367-7_26 Human cell culture processes developed at research laboratory scale need to be translated to large-scale production processes to achieve commercial application to a large market. To allow this transition of scale with consistent process performance and control of costs, it will be necessary to reduce manual processing and increase automation. There are a number of commercially available platforms that will reduce manual process intervention and improve process control for different culture formats. However, in many human cell-based applications, there is currently a need to remain close to the development format, usually adherent culture on cell culture plastic or matrix-coated wells or flasks due to deterioration of cell quality in other environments, such as suspension. This chapter pres... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5398357 Sat, 12 Nov 2011 02:41:18 +0100 5398357 http://www.medworm.com/index.php?rid=5398356&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-367-7_25 Laser microdissection (LMD) microscopy allows isolation of specific cell populations to target their ­molecular profile. There are several different types of LMD microscopes, but they are all based on the same principle. A laser beam is used to cut out cells or tissues of interest from a histological section, cytology preparations, or live cells from tissue cultures. Live cells can be isolated using LMD and processed for downstream molecular work. RNA, DNA, and protein isolation is possible from a small number of cells and the material is suitable for further real-time PCR, ELISA, Western Blotting, and protein microarray analysis. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5398356 Sat, 12 Nov 2011 02:41:18 +0100 5398356 http://www.medworm.com/index.php?rid=5398355&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-367-7_24 With the use of lasers for ablation purposes in spinal surgery, the tissue temperature increases above the boiling point of water, leading to tissue ablation by vaporisation. Due to the thermal environment engendered by the use of lasers, there is concern about the safety of the surrounding important structures, such as dura mater, dorsal root ganglia, and nerve roots. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5398355 Sat, 12 Nov 2011 02:41:18 +0100 5398355 http://www.medworm.com/index.php?rid=5398354&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-367-7_23 Bone remodelling occurs throughout life via the coupled actions of bone resorption and bone formation. When the balance of bone resorption and formation becomes unequal, bone diseases, such as osteoporosis occur, while the absence of functional osteoclasts leads to diseases such as osteopetrosis and pycnodysostosis. In order to develop effective treatments for bone disease the normal regulatory systems involved in bone resorption need to be fully elucidated. The only cell in the body capable of resorbing bone is the osteoclast – a highly specialized cell of haematopoietic origin. Until relatively recently, the ability to study the formation and function of human osteoclasts in vitro has been limited. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5398354 Sat, 12 Nov 2011 02:41:18 +0100 5398354 http://www.medworm.com/index.php?rid=5398353&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-367-7_22 The skeleton is a dynamic organ that is constantly active throughout life. The highly coordinated actions of bone cells early in life determine the body’s shape and form, whilst the constant remodelling (bone resorption followed by an equal amount of bone formation) during adulthood helps to maintain skeletal mass and repair microdamage. When the balance of bone resorption and bone formation becomes unequal, bone diseases, such as osteoporosis, occur. In order to develop drugs to combat bone disease, it is important to know the regulatory systems involved in normal bone formation and resorption. In this chapter, we concentrate on bone formation, providing a detailed guide to isolating and culturing primary human osteoblasts in bone explant cultures, as well as the methodology used to... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5398353 Sat, 12 Nov 2011 02:41:18 +0100 5398353 http://www.medworm.com/index.php?rid=5398352&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-367-7_21 The human adult articular chondrocyte is a unique cell type that has reached a fully differentiated state as an end point of development. Within the cartilage matrix, chondrocytes are normally quiescent and maintain the matrix constituents in a low-turnover state of equilibrium. Isolated chondrocytes in culture have provided useful models to study cellular responses to alterations in the environment such as those occurring in different forms of arthritis. However, expansion of primary chondrocytes in monolayer culture results in the loss of phenotype, particularly if high cell density is not maintained. This chapter describes strategies for maintaining or restoring differentiated phenotype by culture in suspension, gels, or scaffolds. Techniques for assessing phenotype involving primarily ... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5398352 Sat, 12 Nov 2011 02:41:18 +0100 5398352 http://www.medworm.com/index.php?rid=5398351&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-367-7_20 CD4+CD25highCD127low/neg regulatory T cells (Tregs) play a critical role in the maintenance of peripheral tolerance and in controlling the development of autoimmune diseases. A combination of surface and intracellular markers, namely, CD25, CD39/CD73, CD62L, CD45RO, CD127, glucocorticoid-induced tumor necrosis factor receptor (GITR), CTLA-4, and the forkhead/winged helix transcription factor (FOXP3), has been used to characterize Tregs. Tregs suppress T effector responses mainly in a direct cell–cell contact manner. However, other mechanisms independent from this manner cannot be excluded entirely. It has been shown that Tregs can undergo limited expansion in vitro after the stimulation of TCR in the presence of exogenous cytokines, e.g., IL-2. Expanded Tregs retain their suppression... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5398351 Sat, 12 Nov 2011 02:41:18 +0100 5398351 http://www.medworm.com/index.php?rid=5398350&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-367-7_1 The basics of cell culture as applied to human cells are discussed. Biosafety when working with human tissue, which is often pathogenic, is important. The requirements for a tissue culture laboratory are described, particularly the range of equipment needed to carry out cell isolation, purification, and culture. Steps must be taken to maintain aseptic conditions to prevent contamination of cultures with micro-organisms. Basic cell-handling techniques are discussed, including choice of media, primary culture, and cryopreservation of cells so they can be stored for future use. Common assays which are used to determine cell viability and activity are considered. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5398350 Sat, 12 Nov 2011 02:41:18 +0100 5398350 http://www.medworm.com/index.php?rid=5398349&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-367-7_19 Lymphocyte activation and fine tuning of downstream signaling circuits for the regulation of cytokine expression are critical for a successful immune response. Hence, technical protocols permitting simultaneous testing of these attributes in peripheral blood lymphocytes are of paramount importance. Phospho-specific flow cytometry is a novel methodology that detects phosphorylation of signaling effectors in multiple, rare cellular populations within peripheral blood. In addition, it allows the quantification of phosphorylation levels for signaling proteins within each single cell, and therefore is superior compared to traditional biochemical approaches, such as Western blotting. One such important signaling pathway within immune cells is the p38 MAPK pathway involved in the regulation of cy... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5398349 Sat, 12 Nov 2011 02:41:18 +0100 5398349 http://www.medworm.com/index.php?rid=5398348&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-367-7_18 The present protocol offers an economical option for the isolation and culture of human endothelial cells for vascular cell biology research due to the non-invasive collection procedure being devoid of ethical concerns and ease of the isolation technique, expansion and maintenance under standard cell culture conditions. The human umbilical vein endothelial cell (HUVEC) model is useful for any research on general properties of human endothelium, but as these cells are of foetal and venous origin, other sources could be more appropriate models for studies on specific pathological areas, for example, atherosclerosis or cancer angiogenesis. Nevertheless, HUVEC still represent the most simple and available human vascular cell type widely used in biomedical research. (Source: Springer protocols ... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5398348 Sat, 12 Nov 2011 02:41:18 +0100 5398348 http://www.medworm.com/index.php?rid=5398347&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-367-7_17 Human vascular smooth muscle cells (VSMCs) in culture are an important tool in understanding how VSMCs function and contribute to vessel wall contraction as well as disease. In this chapter, we describe methodologies that enable the investigator to culture large numbers of proliferative VSMCs. These VSMCs are heterogeneous and vary in size, shape, and proliferative capacity depending on the disease state and location of the vessel of origin. Therefore, we also describe techniques to validate their identity as bone fide VSMCs. Briefly, the methods include information on how to dissect the blood vessel to remove the medial layer containing VSMCs, as well as methods on how to propagate these cells, by either allowing VSMCs to migrate from the explanted medial tissue or by enzymatically disper... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5398347 Sat, 12 Nov 2011 02:41:18 +0100 5398347 http://www.medworm.com/index.php?rid=5398346&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-367-7_16 Ovary is a polymorphic complex structure in which the cells are arranged in two essential endocrine mini glands: the follicle (F) and the corpus luteum (CL). Their secretory function creates an optimal milieu for growth, maturation, and selection of follicles and oocytes competent for ovulation. Monoculture of isolated ovarian cells has identified the secretory potential of the different cell types functioning in this complex gland in vivo. Primary culture of isolated ovarian cells is a good tool for the investigation of cell interactions and its impact on steroidogenesis, dynamics of steroidogenic enzymes, hormone receptors, changes in the cytoskeleton in granulosa cell populations, regulatory mechanisms, and the intracellular pathways of gonadotropin signaling in steroidogenic ovarian ce... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5398346 Sat, 12 Nov 2011 02:41:18 +0100 5398346 http://www.medworm.com/index.php?rid=5398345&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-367-7_15 Culture of adipose tissue precursor cells allows gaining insight into the sequential processes involved in adipocyte development. Furthermore, the secretory properties associated with these cellular changes can be studied. Although clonal cell lines are valuable tools for the identification of mechanisms associated with proliferation or differentiation such models do not necessarily represent the complexity of adipose tissue physiology. Primary cell culture systems may be closer to physiology and circumvent some of these restrictions. One advantage is that phenotypic properties of the tissue donor such as gender, age, or body weight are still, at least partially retained in vitro. In primary culture, also differences between various adipose depots can be studied either as a condition per s... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5398345 Sat, 12 Nov 2011 02:41:18 +0100 5398345 http://www.medworm.com/index.php?rid=5398344&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-367-7_14 Adipose tissue (AT) is no longer considered merely as insulation or padding for human organs. It is an endocrine organ in its own right, which includes composite cells with the ability to differentiate into multiple cell lines. In fact, there is increasing evidence to support the theory that the causation of obesity and its associated metabolic disorders originate at the cellular or tissue level. Adipocyte dysfunction and chronic inflammatory states are able to modulate triglyceride storage and mobilization directly through cytokine and adipokine release. Significant variability exists between adipocyte isolation and culture techniques which subsequently can impact experimental results. We aim to explain the importance of controlling these variables, to assist tailoring methodological choi... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5398344 Sat, 12 Nov 2011 02:41:18 +0100 5398344 http://www.medworm.com/index.php?rid=5398343&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-367-7_13 Culture of isolated kidney glomerular cells has been employed for almost four decades as a tool to dissect pathophysiological effects of individual cell types in renal disease. This chapter aims to highlight in detail the available techniques to isolate, culture, and characterize human glomerular epithelial and mesangial cells. To establish primary culture of these cells, glomeruli are isolated from the cortex of kidney by differential sieving and cellular outgrowths from cultured glomeruli further subcultured in appropriately coated tissue culture plates/flasks. Methods used for characterization of isolated glomerular mesangial and epithelial cells (podocytes) are described as are the phenotypic markers useful for identification. Other sources of isolated glomerular cells such as immortal... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5398343 Sat, 12 Nov 2011 02:41:18 +0100 5398343 http://www.medworm.com/index.php?rid=5398342&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-367-7_12 Renal physiology and pathology are complex systems that are best studied in whole living organisms. This, however, is often restricted by our desire to limit the number of animal experiments undertaken and to replace them with relevant in vitro models that can be used as surrogates for the system under test. Primary culture cells are derived directly from the relevant tissue and therefore correlate more closely with the system under examination. Although the tissue of origin is not always readily available for culture and cells may quickly change their phenotype after only a few passages, they can be used in many circumstances to validate results obtained from closely related cell lines and to confirm vital protein expression patterns. This chapter outlines methods by which proximal tubula... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5398342 Sat, 12 Nov 2011 02:41:18 +0100 5398342 http://www.medworm.com/index.php?rid=5398341&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-367-7_11 The intestinal epithelium is a highly dynamic tissue undergoing constant and rapid renewal. It consists of a functional villus compartment responsible for terminal digestion and nutrient absorption and a progenitor cell compartment located in the crypts that produce new cells. The mechanisms regulating cell proliferation in the crypt, their migration, and differentiation are still incompletely understood. Until recently, normal human intestinal cell models allowing the study of these mechanisms have been lacking. In our laboratory, using fetal human intestines obtained at mid-gestation, we have generated the first normal human intestinal epithelial crypt-like (HIEC) cell line and villus-like primary cultures of differentiated enterocytes (PCDE). In this chapter, we provide a detailed descr... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5398341 Sat, 12 Nov 2011 02:41:18 +0100 5398341 http://www.medworm.com/index.php?rid=5398340&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-367-7_10 Our understanding of gastric epithelial physiology in man is limited by the absence of normal or appropriate cancer cell lines that could serve as an in vitro model. Research mostly relied on primary culture of gastric epithelial cells of animal species, enriched with surface mucous cells, and devoid of glandular zymogenic chief cells. We successfully applied a new nonenzymatic procedure using Matrisperse Cell Recovery Solution to dissociate the entire epithelium from human fetal stomach. Cultures were generated by seeding multicellular aggregates prepared by mechanical fragmentation. We further demonstrate that this simple and convenient technique allows for the maintenance of heterogenous gastric epithelial primary cultures on plastic without a biological matrix as well as the persistenc... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5398340 Sat, 12 Nov 2011 02:41:18 +0100 5398340 http://www.medworm.com/index.php?rid=5158328&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-273-1_9 Anti-cancer therapy is largely comprised of radiation, surgery, and chemotherapy treatments. Although a single mode of therapy can be effective in treating certain types of cancer, none presents a cure. Multi-modal therapy, the use of two or more agents in combination (e.g., radiation and chemotherapy together), shows potential for a more effective treatment of cancer. The challenge then is identifying effective therapy combinations. In this chapter, we describe the use of Drosophila as a whole animal in vivo model to screen for small molecules that effectively combine with ionizing radiation to kill checkpoint mutants preferentially over wild-type. The differential use of wild-type and checkpoint mutants has the potential to identify molecules that act in a genotype-specific manner to era... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5158328 Fri, 26 Aug 2011 13:13:45 +0100 5158328 http://www.medworm.com/index.php?rid=5158327&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-273-1_8 Under genotoxic stress, activation of cell cycle checkpoint responses leads to cell cycle arrest, which allows cells to repair DNA damage before continuing to cycle. Drosophila larval epithelial sacs, called imaginal discs, are an excellent in vivo model system for studying radiation-induced cell cycle arrest. Larval imaginal discs go into cell cycle arrest after being subjected to low-dose irradiation, are subject to easy genetic manipulation, are not crucial for survival of the organism, and can be dissected easily for further molecular or cellular analysis. In this chapter, we describe methods for assessing low-dose irradiation-induced cell cycle arrest. Mitotic cells are identified by immunofluorescence staining for the mitotic marker phosphorylated histone H3 (phospho-histone H3 or pH... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5158327 Fri, 26 Aug 2011 13:13:44 +0100 5158327 http://www.medworm.com/index.php?rid=5158326&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-273-1_7 Live-imaging of cells has been an excellent technique to provide us with highly accurate and valuable information about cell cycle checkpoint regulation and DNA damage responses. Early stage Drosophila embryos have several advantages to be studied by live-imaging. Fly embryos are much tougher than cultured cells and stand up to relatively rough manipulation, such as protein/chemical microinjection followed by time-lapse imaging. Cell cycles in the embryonic cleavage stage progress rapidly (9–20 min/cycle) and nuclear divisions are synchronous, allowing observation of multiple nuclei/cell cycles in a short period of time. Somatic precursor nuclei form a monolayer at the cortex of the embryo during the syncytial blastoderm stage (cell cycles 10–13). Thus the nuclei in this s... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5158326 Fri, 26 Aug 2011 13:13:44 +0100 5158326 http://www.medworm.com/index.php?rid=5158325&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-273-1_6 Drosophila cell lines are valuable tools to study a number of cellular processes, including DNA damage responses and cell cycle checkpoint control. Using an in vitro system instead of a whole organism has two main advantages: it saves time and simple and effective molecular techniques are available. It has been shown that Drosophila cells, similarly to mammalian cells, display cell cycle checkpoint pathways required to survive DNA damaging events (de Vries et al. 2005, Journal of Cell Science 118, 1833–1842; Bae et al. 1995, Experimental Cell Research 217, 541–545). Moreover, a number of proteins involved in checkpoint and cell cycle control in mammals are highly conserved among different species, including Drosophila (de Vries et al. 2005, Journal of Cell Science... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5158325 Fri, 26 Aug 2011 13:13:44 +0100 5158325 http://www.medworm.com/index.php?rid=5158324&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-273-1_5 Methods are described here to monitor changes in protein level and subcellular localization during the cell cycle progression in the budding yeast Saccharomyces cerevisiae. Cell synchronization is achieved by an α-factor-mediated block-and-release protocol. Cells are collected at different time points for the first two cell cycles upon release. Cellular DNA contents are analyzed by flow cytometry. Trichloroacetic acid protein precipitates are prepared for monitoring levels of cell cycle regulated proteins by Western blotting. The dynamic changes in protein subcellular localization patterns are examined by indirect immunofluorescence microscopy. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5158324 Fri, 26 Aug 2011 13:13:44 +0100 5158324 http://www.medworm.com/index.php?rid=5158323&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-273-1_4 The spindle assembly checkpoint (SAC) is a quality control mechanism for overseeing the fidelity of chromosome segregation. By modulating the activity of the anaphase-promoting complex or cyclosome (APC/C), the SAC sets the timing of anaphase-onset by co-ordinating the timely destruction of key proteins with the completion of chromosome alignment. How mammalian oocytes regulate chromosome segregation during the first meiotic division (meiosis I) is of immense importance as mis-segregation at this crucial stage in human oocytes underpins the majority of human aneuploidy and birth defects. In recent years, the SAC has been shown to be indispensable for the accuracy of meiosis I chromosome segregation. Here, I describe a technique based on immunoblotting for evaluating SAC competence during m... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5158323 Fri, 26 Aug 2011 13:13:44 +0100 5158323 http://www.medworm.com/index.php?rid=5158322&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-273-1_3 In response to post-replicative DNA damage, cells activate the G2 DNA damage checkpoint to ensure mitosis is not attempted until the damage has been repaired. This is a common response to a variety of DNA damaging agents, including ionizing radiation and many chemotherapeutic agents used in the treatment of cancer. The G2 DNA damage checkpoint acts to inhibit the mitotic cyclin-dependent kinase, and thus cells are arrested in the G2 phase of the cell cycle. The kinetics of this checkpoint can be assayed by staining cells for markers of mitosis, which can then be quantified by flow cytometry or microscopy. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5158322 Fri, 26 Aug 2011 13:13:44 +0100 5158322 http://www.medworm.com/index.php?rid=5158321&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-273-1_2 Slowing of replication in response to DNA damage is a universal response to DNA damage during S-phase. Originally discovered to be defective in checkpoint mutant cells in metazoans, this S-phase DNA damage checkpoint response has been extensively studied in yeast. Unlike other checkpoints that completely arrest cell cycle, the S-phase DNA damage checkpoint slows but does not completely halt replication in response to DNA damage. An analysis of mutants defective in the slowing response requires a sensitive assay to measure this quantitative effect. The use of centrifugal elutriation to synchronize cells and improved techniques in preparing cells for flow cytometry allow for more sensitive and accurate measurement of cells’ ability to slow replication in the presence of DNA damage. Thi... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5158321 Fri, 26 Aug 2011 13:13:44 +0100 5158321 http://www.medworm.com/index.php?rid=5158320&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-273-1_1 We present a detailed explanation of the setup and use of centrifugal elutriation to synchronize cells in G2, exposure of cells to DNA damage, and measurement of mitotic progression and delay. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5158320 Fri, 26 Aug 2011 13:13:44 +0100 5158320 http://www.medworm.com/index.php?rid=5158319&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-273-1_19 Cell cycle checkpoints operating through a network of multiple signaling pathways provide a key mechanism for self-defense of cells against DNA damage caused by various endogenous or environmental stresses. In cancer treatment, checkpoints are activated in response to diverse DNA-damaging agents and radiation, thus representing a critical barrier limiting therapeutic efficacy. To date, despite efforts to target other components of checkpoint signaling pathways (e.g., ATM, Chk2, Wee1), checkpoint kinase 1 (Chk1) remains the most important target for cancer treatment because of its functional association with essentially all cell cycle checkpoints. The primary goal in the development of therapeutic agents targeting cell cycle checkpoints continues to be improving the anti-cancer activity of ... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5158319 Fri, 26 Aug 2011 13:13:44 +0100 5158319 http://www.medworm.com/index.php?rid=5158318&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-273-1_18 DNA interstrand crosslinks (ICLs) are lesions that covalently link the two strands of DNA. This type of DNA damage represents one of the most complex DNA lesions whose repair mechanisms remain largely unclear. Uncovering proteins involved in the processing of ICLs and understand how they interact with the damaged DNA in vivo is crucial for the understanding of DNA interstrand crosslink repair processes. Moreover, the presence of an ICL during S phase constitutes the most severe blockage to DNA synthesis and results in prolonged stall of replication forks. The mechanisms of resolving a stalled replication fork is poorly understood because proper experimental platforms are lacking. To enable detection of protein recruitment to site-specific ICLs and to ICL-stalled replication forks, we estab... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5158318 Fri, 26 Aug 2011 13:13:43 +0100 5158318 http://www.medworm.com/index.php?rid=5158317&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-273-1_17 In yeast like all eukaryotes, microtubules are a crucial element of the mitotic spindle that separates the genetic material during cell division. The assembly status and position of the mitotic spindle, as well as cytoplasmic microtubules, can be monitored easily using indirect immunofluorescence with antibodies against tubulin. A detailed protocol is described for Saccharomyces cerevisiae that involves the fixation of actively growing cells, removal of the cell wall by enzymatic digestion, post-fixation, and the application of tubulin antibodies. The use of secondary antibodies conjugated to a fluorescent moiety permit visualization of the mitotic spindle by fluorescence microscopy. Methods for the reduction of background and pre-absorption of antibodies are discussed. (Source: Springer p... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5158317 Fri, 26 Aug 2011 13:13:43 +0100 5158317 http://www.medworm.com/index.php?rid=5158316&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-273-1_16 Maintenance of genomic integrity is critical for the survival of organisms. Thus, mammalian cells employ a complex DNA damage response that can sense and repair DNA damage. One important aspect of the cellular DNA damage response is the activation of checkpoints that result in cell cycle arrest. In this chapter we present methods for the induction of genotoxic stress. Additionally, we describe methods for studying the progression of cells from G1 to S phase after genotoxic stress. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5158316 Fri, 26 Aug 2011 13:13:43 +0100 5158316 http://www.medworm.com/index.php?rid=5158315&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-273-1_15 Cell proliferation depends on the timely synthesis and destruction of proteins at specific phases of the cell cycle. Recently it was discovered that the destruction of several key cell cycle regulatory proteins during S phase is coupled directly to DNA replication. These proteins harbor a motif called a PIP degron that mediates binding to chromatin bound PCNA at replication forks and recruits the CRL4Cdt2 E3 ubiquitin ligase. These interactions comprise an elegant mechanism for coupling DNA replication with ubiquitylation and subsequent proteolysis by the 26S proteasome. Here we describe a flow cytometry-based method using Drosophila S2 cells that recapitulates S phase-specific protein proteolysis. Because of the high degree of evolutionary conservation of the PIP degron and CRL4Cdt2 and t... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5158315 Fri, 26 Aug 2011 13:13:43 +0100 5158315 http://www.medworm.com/index.php?rid=5158314&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-273-1_14 The most critical feature of the cellular response to DNA damage is the ability of the cell to pause and repair the damage so that detrimental mutations will not be passed along to future generations of cells. The cell cycle of mammalian cells is equipped with checkpoints that can prevent cell cycle progression. Cells can either be arrested before replication of the DNA when the cells have a 2 N amount of DNA or after replication and prior to cell mitosis when the cells have a 4 N amount of DNA. Flow cytometry is a standard technique that is used to ‘sort’ cells based on their DNA content. It uses the principles of light scattering, light excitation, and emission of fluorochrome molecules to generate data about individual cells. The cells are fixed and permeabilized s... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5158314 Fri, 26 Aug 2011 13:13:43 +0100 5158314 http://www.medworm.com/index.php?rid=5158313&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-273-1_13 The Ataxia telangiectasia-mutated (ATM) and the ATM-Rad3-related (ATR) kinases are master regulators of the DNA damage-signaling pathways that respond to a wide variety of DNA damage. In this chapter, we describe an in vitro biochemical assay to study the activation of ATM and ATR by double-stranded DNA breaks (DSBs) (Shiotani and Zou, 2009, Mol Cell 33, 547–58). In this assay, DNA fragments with different structural features are used to activate ATM and ATR in human cell extracts, and the activation of ATM and ATR is monitored by the phosphorylation of specific ATM and ATR substrates. Importantly, in this assay both ATM and ATR are activated in a DNA structure-regulated manner, providing a useful tool to characterize the DNA structural determinants for their activation. The f... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5158313 Fri, 26 Aug 2011 13:13:43 +0100 5158313 http://www.medworm.com/index.php?rid=5158312&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-273-1_12 Attempts to passage through mitosis with unrepaired DNA damage or incompletely replicated DNA leads to genome instability and/or cell death. To prevent this from occurring, an ancient checkpoint (known as the G2 DNA damage checkpoint) that inhibits the activation of the mitotic cyclin-dependent kinase is activated to hold cells in the G2 phase of the cell cycle. The effector of this checkpoint is Chk1, a protein serine-threonine kinase. Chk1 contains an N-terminal catalytic domain, and C-terminal regulatory domain. Within the regulatory domain there are two residues, Serine-317 (S317) and Serine-345 (S345), which are phosphorylated in active Chk1 molecules, and subsequently dephosphorylated to inactivate Chk1 and allow mitotic entry. Phospho-specific antibodies can be used to detect these ... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5158312 Fri, 26 Aug 2011 13:13:43 +0100 5158312 http://www.medworm.com/index.php?rid=5158311&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-273-1_11 Following acquisition of DNA damage S-phase progression may potentially be affected via multiple mechanisms. For example DNA damage-activated signal transduction pathways negatively regulate the initiation of DNA synthesis at unfired origins of replication, a process termed the ‘S-phase checkpoint’ or the ‘intra-S-phase checkpoint’. Additionally, many DNA lesions pose physical barriers to replication forks and therefore inhibit DNA synthesis directly by blocking the elongation of active replicons. Inhibition of DNA synthesis in response to DNA damage is commonly assayed by measuring incorporation of radiolabeled or halogenated nucleotides into bulk genomic DNA. However, these techniques do not distinguish between effects of DNA damage on initiation and elongation ph... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5158311 Fri, 26 Aug 2011 13:13:42 +0100 5158311 http://www.medworm.com/index.php?rid=5158310&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-273-1_10 Cell cycle checkpoints are involved in the coordinated response to DNA damage and thus play a key role in maintaining genome integrity. Several model systems have been developed to study the mechanisms and complexity of checkpoint function. Here we describe the application of cell-free extracts derived from Xenopus eggs as a model system to investigate DNA replication, damage, and checkpoint activation. We outline the preparation of cell-free extracts, DNA substrates and their subsequent use in assays aimed at understanding cell cycle checkpoints, and related processes. Several advances made over the years have enabled the continued use of the Xenopus system to answer a variety of questions in DNA replication, repair and checkpoint signaling. It is anticipated that the versatile Xenopus sy... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5158310 Fri, 26 Aug 2011 13:13:42 +0100 5158310 http://www.medworm.com/index.php?rid=5085853&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-210-6_13 Nonhuman primates (NHP) are the closest animal species to humans and have been widely used for studying human reproductive physiology. Assisted reproductive technology (ART) in Old World NHPs provides great opportunity for studying fertilization, embryo development, embryonic stem cell (ESC) derivation for regenerative medicine, somatic cell nuclear transfer (cloning), and transgenic NHP models of inherited genetic disorders. Here we present two ART protocols developed for rhesus monkey (Macaca mulatta) and baboon (Papio cynocephalus). (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5085853 Tue, 02 Aug 2011 08:31:28 +0100 5085853 http://www.medworm.com/index.php?rid=5085852&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-210-6_12 One of the most straightforward approaches to making novel biological discoveries is the forward genetic screen. The time is ripe for forward genetic screens in the mouse since the mouse genome is sequenced, but the function of many of the genes remains unknown. Today, with careful planning, such screens are within the reach of even small individual labs. In this chapter we first discuss the types of screens in existence, as well as how to design a screen to recover mutations that are relevant to the interests of a lab. We then describe how to create mutations using the chemical N-ethyl-N-nitrosourea (ENU), including a detailed injection protocol. Next, we outline breeding schemes to establish mutant lines for each type of screen. Finally, we explain how to map mutations using recombinatio... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5085852 Tue, 02 Aug 2011 08:31:27 +0100 5085852 http://www.medworm.com/index.php?rid=5085851&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-210-6_11 Establishment of methods to inactivate genes by homologous recombination in embryonic stem (ES) cells has provided great advantages to the field of mouse genetics. Using this technology, a number of null mutant mice, so-called knock-out mice, have been generated. The gene-targeting technology offers a strong tool that allows us to understand the function of a particular gene of interest in the whole animal and has contributed to studies in a wide variety of biological research areas. More recently, the original knock-out technology has been further refined to develop advanced strategies to generate conditional knock-out and knock-in mice. In this chapter, an overview of gene-targeting strategies is presented and procedures to generate these genetically engineered mice are discussed. (Sourc... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5085851 Tue, 02 Aug 2011 08:31:27 +0100 5085851 http://www.medworm.com/index.php?rid=5085850&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-210-6_10 Analysis of gene expression patterns is central to the study of embryonic development. This chapter details methods for detecting gene expression in whole mouse embryos and in tissue sections. The most commonly used methods available in mouse are described and include mRNA in situ hybridization, immunohistochemistry, and detection of enzymatic and fluorescent protein reporters. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5085850 Tue, 02 Aug 2011 08:31:27 +0100 5085850 http://www.medworm.com/index.php?rid=5061971&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-252-6_9 Assaying microtubule dynamics in vitro requires stabilized nucleation centers, a method to immobilize individual microtubules onto a surface, and a specialized microscope to image the microtubule. Microtubules are polar structures with different dynamic properties at the plus and minus ends. However, the dynamics of the two ends can be modified by the addition of other proteins, such as microtubule plus-end-tracking proteins (+TIPs), so that it becomes impossible to distinguish the microtubule polarity by measuring the differences in the dynamic properties of the ends alone. In this chapter, we describe a method for labeling tubulin protein with N-hydroxysuccinimide ester fluorescent dyes, enabling the formation of dual-color polarity-marked stable microtubule seeds that can be immobilized... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5061971 Mon, 25 Jul 2011 22:12:36 +0100 5061971 http://www.medworm.com/index.php?rid=5061970&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-252-6_8 The centrosome is a unique organelle that nucleates and organizes the interphase microtubule array and facilitates bipolar spindle assembly during mitosis. Isolated centrosomes are ideal starting materials for biochemical and structural studies as well as for functional assays to study microtubule-dependent processes. In fact, they are hitherto the only system to study radial microtubule arrays in vitro. This chapter provides a practical method and the rationale of isolating centrosomes from adherent cultured cells by density-gradient centrifugation. It further describes how to evaluate the activity and function of centrosomes by microtubule nucleation and immunofluorescence assays and how to measure microtubule dynamics nucleated from isolated centrosomes in Xenopus egg extracts. (Source:... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5061970 Mon, 25 Jul 2011 22:12:36 +0100 5061970 http://www.medworm.com/index.php?rid=5061969&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-252-6_7 Turbidity measurements are rapid and reliable methods to follow the effects of drugs, proteins, nucleotides, metals, and other factors on the assembly kinetics of tubulin into microtubules in vitro and have been in use since 1974. Improvements in spectrophotometers and software have resulted in the use of less protein, and more curves can be analyzed continuously and almost simultaneously. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5061969 Mon, 25 Jul 2011 22:12:35 +0100 5061969 http://www.medworm.com/index.php?rid=5061968&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-252-6_6 Dynamic instability is a hallmark of the microtubule cytoskeleton. In order to regulate microtubule dynamics in vivo, a varied host of microtubule-associated proteins are mobilized to promote microtubule nucleation, growth, stabilization, catastrophe, depolymerization, rescue, and severing. To confer these various functions, cytoskeletal regulators have highly tuned affinities for tubulin, recognizing the unpolymerized αβ-tubulin heterodimer, dynamic microtubule lattice, stabilized microtubule lattice, or a combination therein. The protocols presented here probe αβ-tubulin and microtubule binding using gel filtration and cosedimentation, respectively. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5061968 Mon, 25 Jul 2011 22:12:35 +0100 5061968 http://www.medworm.com/index.php?rid=5061967&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-252-6_5 Alpha tubulin comprises a C-terminal tyrosine residue, which is subject to cyclic removal from the peptide chain by a still uncharacterized carboxypeptidase and re-addition to the chain by a tubulin tyrosine ligase. We have shown in different animal or human models that the presence or absence of the tyrosine residue had dramatic consequences for both tumor progression and neuronal organization. In cells, tubulin detyrosination impairs the proper localization of CAP-Gly proteins at microtubule + end, compromises the activity of microtubule-depolymerizing motors of the Kinesin 13 family, and favors both spastin microtubule-severing activity and kinesin 1 processivity. The biochemical basis for these cellular effects of tubulin detyrosination can now be investigated in reconstituted systems ... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5061967 Mon, 25 Jul 2011 22:12:35 +0100 5061967 http://www.medworm.com/index.php?rid=5061966&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-252-6_4 Microtubules are cytoskeletal structures built of alpha- and beta-tubulins. Although tubulins are highly conserved throughout evolution, microtubules can be adapted to a range of different functions. A powerful mechanism that could regulate the functional specialization of microtubules is the posttranslational modification of tubulin molecules. Two tubulin modifications, polyglutamylation and polyglycylation, generate amino acid side chains of different length on tubulin. These modifications are thought to regulate interactions between microtubules and their associated proteins; however, detailed studies of this potential mechanism have not been performed. The investigation of the potential regulatory role of polyglutamylation requires in vitro tools to visualize the molecular events that ... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5061966 Mon, 25 Jul 2011 22:12:35 +0100 5061966 http://www.medworm.com/index.php?rid=5061965&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-252-6_3 We describe the preparation of milligram quantities of highly purified native tubulin from S. pombe suitable for use in microtubule dynamics assays as well as structural and other biochemical studies. S. pombe cells are grown in bulk in a fermenter and then lysed using a bead mill. The soluble protein fraction is bound to anion-exchange chromatography resin by batch binding, packed in a ­chromatography column and eluted by a salt gradient. The tubulin-containing fraction is ammonium sulphate precipitated to further concentrate and purify the protein. A round of high-resolution anion-exchange chromatography is carried out before a cycle of polymerisation and depolymerisation to select functional tubulin. Gel filtration is used to remove residual contaminants before a final desalting ste... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5061965 Mon, 25 Jul 2011 22:12:35 +0100 5061965 http://www.medworm.com/index.php?rid=5061964&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-252-6_2 Microtubules, polymers of the heterodimeric protein αβ-tubulin, give shape to cells and are the tracks for vesicle transport and chromosome segregation. In vitro assays to study microtubule functions and their regulation by microtubule-associated proteins require the availability of purified αβ-tubulin. In this chapter, we describe the process of purification of heterodimeric αβ-tubulin from porcine brain. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5061964 Mon, 25 Jul 2011 22:12:35 +0100 5061964 http://www.medworm.com/index.php?rid=5061963&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-252-6_21 Microtubule organization and dynamics are controlled by a large set of cellular factors. An important group of microtubule regulators is microtubule plus-end-tracking proteins (+TIPs), which accumulate specifically at the growing microtubule ends, affect different phases of dynamic instability, and link microtubules to various cellular structures. +TIPs include a very diverse set of proteins with widely different structural properties. One of the most conserved and ubiquitous +TIP families are end-binding (EB) proteins, which can track growing microtubule ends autonomously in the absence of any other factors. In contrast, the majority of other known +TIPs cannot recognize the growing microtubule plus ends on their own; instead, they “hitchhike” to the plus ends by interacting w... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5061963 Mon, 25 Jul 2011 22:12:34 +0100 5061963 http://www.medworm.com/index.php?rid=5061962&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-252-6_20 The microtubule (MT) cytoskeleton is required for many aspects of cell function, including the transport of intracellular materials, maintenance of cell polarity, and the regulation of mitosis. These functions are coordinated by MT-associated proteins (MAPs), which work in concert with each other, binding MTs and altering their properties. We have used an MT co-sedimentation assay, combined with 1D and 2D PAGE and mass spectrometry, to isolate MAPs from early Drosophila embryos. This technique has identified many novel proteins and an association with MTs for many known proteins, previously not described as associating with MTs. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5061962 Mon, 25 Jul 2011 22:12:34 +0100 5061962 http://www.medworm.com/index.php?rid=5061961&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-252-6_1 Microtubules are one of the most spectacular features in the cell: long, fairly rigid tubules that provide physical strength while at the same time serving as tracks of the intracellular transport network. In addition, they are the main constituents of the cell division machinery, and guide axonal growth and the direction of cell migration. To be able to fulfil such diverse functions, microtubules have to be arranged into suitable patterns and remodelled according to extra- and intracellular cues. Moreover, the delicate regulation of microtubule dynamics and the dynamic interactions with subcellular structures, such as kinetochores or cell adhesion sites, appear to be of crucial importance to microtubule functions. It is, therefore, important to understand microtubule dynamics and its spat... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5061961 Mon, 25 Jul 2011 22:12:34 +0100 5061961 http://www.medworm.com/index.php?rid=5061960&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-252-6_19 Laser ablation is a powerful tool that can be used to study a variety of biological mechanisms. Microscopes with high optical performances are nowadays available, and lasers that could be used to perform ablations have become accessible to every laboratory. Setting up a laser ablation system is a relatively straightforward task; however, it requires some basic knowledge of optics. We illustrate the fundamental components of the experimental setup and describe the most common pitfalls and difficulties encountered when designing, setting up, and working with a laser ablation system. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5061960 Mon, 25 Jul 2011 22:12:34 +0100 5061960 http://www.medworm.com/index.php?rid=5061959&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-252-6_18 This chapter presents protocols not only used to investigate the effect of microtubule-targeting agents on microtubule dynamic instability parameters, but also their impact on loading +TIPs at microtubule plus ends. These agents can be considered either as drugs to analyze their pharmacological effects on microtubule dynamics and their subsequent functions or as tools to improve basic knowledge on the regulation of microtubule dynamics. Deciphering the complexes of proteins that regulate microtubule dynamic instability may lead to the discovery of new potential targets for therapy. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5061959 Mon, 25 Jul 2011 22:12:34 +0100 5061959 http://www.medworm.com/index.php?rid=5061958&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-252-6_17 Cell polarization is important for multiple physiological processes. In polarized cells, microtubules (MTs) are organized into a spatially polarized array. Generally, in nondifferentiated cells, it is assumed that MTs are symmetrically nucleated exclusively from centrosome [microtubule organizing center (MTOC)] and then reorganized into the asymmetric array. We have recently identified the Golgi complex as an additional MTOC that asymmetrically nucleates MTs toward one side of the cell. Methods used for alternative MTOC identification include microtubule regrowth after complete drug-induced depolymerization and tracking of growing microtubules using fluorescently labeled MT +TIP binding proteins in living cells. These approaches can be used for quantification of MT nucleation sites at dive... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5061958 Mon, 25 Jul 2011 22:12:33 +0100 5061958 http://www.medworm.com/index.php?rid=5061957&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-252-6_16 The development of photactivatable (PA) variants of Green fluorescent protein (GFP) has allowed the dynamics of spatially restricted protein pools within living cells to be determined. Over the last 5 years, experiments utilizing PA-GFP fused to α-tubulin have provided important insights into the mechanisms that control microtubule dynamics in living cells. In this chapter, we describe the methodology required to generate stable cell lines expressing photoactivatable-GFP-α-tubulin and to derive quantitative measurements of tubulin turnover at microtubules plus-ends in living cells. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5061957 Mon, 25 Jul 2011 22:12:33 +0100 5061957 http://www.medworm.com/index.php?rid=5061956&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-252-6_15 The movement of chromosomes in mitosis requires spindle microtubules, as well as a set of specific motor proteins located at the kinetochores of the chromosomes. The exact mechanisms of chromosome movement have remained ambiguous for many years. Cumulating evidence indicates that chromosome movement in early mitosis occurs by lateral sliding of kinetochores along the surface of microtubules. We provide here the protocol for an immunological staining method of microtubules that allows electron microscopic analysis of spindle microtubules over a long distance and that has helped clarifying this biological question. The technique involves the use of ultra-small immunogold, enhanced by silver. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5061956 Mon, 25 Jul 2011 22:12:33 +0100 5061956 http://www.medworm.com/index.php?rid=5061955&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-252-6_14 Cryo-electron tomography of vitrified specimens allows visualization of thin biological samples in three-dimensions. This method can be applied to study the interaction of proteins that show disorder and/or bind in a nonregular fashion to microtubules. Here, we describe the protocols we use to observe microtubules assembled in vitro in the presence of XMAP215, a large and flexible protein that binds to discrete sites on the microtubule lattice. Gold particles are added to the mix before vitrification to facilitate image acquisition in low-dose mode and their subsequent alignment before tomographic reconstruction. Three-dimensional reconstructions are performed using the IMOD software, processed with ImageJ and visualized in UCSF Chimera. Extraction of features of interest is performed usin... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5061955 Mon, 25 Jul 2011 22:12:33 +0100 5061955 http://www.medworm.com/index.php?rid=5061954&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-252-6_13 Proteins of the kinesin superfamily share a conserved motor domain, which both hydrolyses adenosine-5′-triphosphate (ATP) and binds microtubules. To determine the mechanism of action of a kinesin, it is necessary to relate the chemical cycle of ATP turnover to the mechanics of microtubule interaction. In this chapter, a number of methods are outlined by which the ATP turnover cycle of a kinesin can be analysed with a particular focus on the use of fluorescently labelled ATP and ADP analogues as a means of isolating individual steps in the cycle. By analysing the ATP turnover cycle of a kinesin, both in solution and in the presence of microtubules, the change in nucleotide state triggered upon microtubule binding can be determined. This provides information vital to understanding the ... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5061954 Mon, 25 Jul 2011 22:12:32 +0100 5061954 http://www.medworm.com/index.php?rid=5061953&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-252-6_12 The direct observation of single kinesins and microtubule-associated proteins (MAPs) has become a core tool for cytoskeleton research. We outline several variations to the core experiment that allow the researcher to explore structural and biophysical mechanisms underlying kinesin motility and MAP function. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5061953 Mon, 25 Jul 2011 22:12:32 +0100 5061953 http://www.medworm.com/index.php?rid=5061952&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-252-6_11 We describe three in vitro assays that can be used to mimic MT interactions with the cell boundary. The essential components in each of our minimal systems are (functionalized) microfabricated barriers against which we grow MTs under different conditions. We describe in detail the different methods and assays necessary to realize these in vitro experiments. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5061952 Mon, 25 Jul 2011 22:12:32 +0100 5061952 http://www.medworm.com/index.php?rid=5061951&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-252-6_10 Several microtubule-associated proteins localize in living cells selectively to an extended region at the growing microtubule plus ends. Over the last years, these plus-end-tracking proteins, also called +TIPs, have attracted considerable interest because they are involved in a large variety of essential intracellular processes. GFP-labeled versions of EB proteins are also often used as markers for intracellular microtubule organization and dynamics. The mechanism of selective +TIP binding to the end region of growing microtubule was unkown. Recently, the phenomenon of end tracking was reconstituted in vitro from purified proteins, which allowed the identification of EB proteins as the minimal core of the plus-end-tracking system and the dissection of the molecular mechanism of end trackin... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5061951 Mon, 25 Jul 2011 22:12:30 +0100 5061951 http://www.medworm.com/index.php?rid=5027571&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-207-6_29 Cell migration, essential in cancer progression, is a complex process comprising a number of spatiotemporally regulated and well-coordinated mechanisms. In order to study (random) cell migration in the context of responses to various external cues (such as growth factors) or intrinsic cell signaling, a number of different tools and approaches have been developed. In order to unravel the key pathways and players involved in the regulation of (cancer) cell migration, a systematical mapping of the players/pathways is required. For this purpose, we developed a cell migration assay based on automatic high-throughput microscopy screen. This approach allows for screening of hundreds of genes, e.g., those encoding various kinases and phosphatases but can also be used for screening of drugs librari... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5027571 Fri, 15 Jul 2011 01:29:56 +0100 5027571 http://www.medworm.com/index.php?rid=5027570&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-207-6_7 The Boyden chamber, initially designed to study leukocyte chemotaxis, has become one of the most used tools to assess cell motility and invasion. The classical Boyden chamber consists of two compartments separated by a membrane representing a physical barrier that cells can overcome only by active migration. Since its initial introduction, a number of different Boyden chamber devices have been developed. The Boyden chamber can be adapted to study tumour cells’ invasive properties by coating the membrane with different extracellular matrix proteins. The method described in this chapter is intended specifically for measuring the migration or invasion of human endothelial and cancer cells. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5027570 Fri, 15 Jul 2011 01:29:56 +0100 5027570 http://www.medworm.com/index.php?rid=5027569&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-207-6_8 The need to identify inhibitors of cancer invasion has driven the development of quantitative in vitro invasion assays. The most common assays used are based on the original Boyden assay system. Today commercially available plastic inserts for multi-well plates, which possess a cell-permeable membrane, as typified by Transwell® Permeable Supports, permit accurate repeatable invasion assays. When placed in the well of a multi-well tissue culture plate these inserts create a two-chamber system separated by the cell-permeable membrane. To create an invasion assay the pores in the membrane are blocked with a gel composed of extracellular matrix that is meant to mimic the typical matrices that tumour cells encounter during the invasion process in vivo. By placing the cells on one side of th... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5027569 Fri, 15 Jul 2011 01:29:55 +0100 5027569 http://www.medworm.com/index.php?rid=5027568&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-207-6_9 Invasive cell migration is critical for leukocyte trafficking into tissues. Podosomes are matrix-degrading adhesive structures that are formed by macrophages and are necessary for macrophage migration and invasion. Here, we describe methods for imaging and quantifying podosomes in primary human macrophages and in THP-1 cells, a monocyte cell line that can be differentiated to a macrophage-like state. Moreover, we outline detailed methods for live imaging of podosomes, which are highly dynamic, and for the quantification of rates of podosome turnover. Finally, we discuss methods for the quantitative analysis of matrix degradation on fluorescent-gelatin-coated cover slips. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5027568 Fri, 15 Jul 2011 01:29:55 +0100 5027568 http://www.medworm.com/index.php?rid=5019064&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-207-6_6 The neuronal growth cone, a highly motile structure at the tip of neuronal processes, is an excellent model system for studying directional cell movements. While biochemical and genetic approaches unveiled molecular interactions between ligand, receptor, signaling, and cytoskeleton-associated proteins controlling axonal growth and guidance, in vitro live cell imaging has emerged as a crucial approach for dissecting cellular mechanisms of growth cone motility and guidance. Important insights into these mechanisms have been gained from studies using the large growth cones elaborated by Aplysia californica neurons, an outstanding model system for live cell imaging for a number of reasons. Identified neurons can be isolated and imaged at room temperature. Aplysia growth cones are five to ten t... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5019064 Wed, 13 Jul 2011 01:02:39 +0100 5019064 http://www.medworm.com/index.php?rid=5019063&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-207-6_5 Much of what is known about the mechanisms regulating cell adhesion and migration come from in vitro studies of cells plated on 2-dimensional (2D) extracellular matrix (ECM) proteins. The importance of studying these processes in cells within 3-dimensional (3D) environments is becoming increasingly recognised, as a number of studies have now demonstrated that adhesion formation and migration are markedly different in cells within 3D environments (Cukierman et al. Science 1708–1712, 2001). It is also known that the composition and pliability or density of the ECM are important in regulating cell adhesion and migration (Cukierman et al. Science 1708–1712, 2001). Cell-derived matrices (CDM) are naturally deposited fibrillar ECM from fibroblasts that can be used to study the adhesi... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5019063 Wed, 13 Jul 2011 01:02:39 +0100 5019063 http://www.medworm.com/index.php?rid=5019062&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-207-6_4 The directed migration of cells (chemotaxis) occurs not only during wound healing and inflammatory responses but also during embryonic development. However, the intracellular signaling pathways that enable a cell to detect a chemoattractant and subsequently migrate toward the source are not clearly defined. The Dunn chemotaxis chamber in conjunction with time-lapse microscopy is a powerful tool that enables the user to observe directly the morphological response of cells to a chemoattractant in real time. Here, using the Dunn chemotaxis chamber, we describe the response of murine bone marrow-derived macrophages to colony stimulating factor-1. This is a particularly useful protocol as it can be adapted to study bone marrow-derived macrophages isolated from genetically modified mice and thus... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5019062 Wed, 13 Jul 2011 01:02:39 +0100 5019062 http://www.medworm.com/index.php?rid=5019061&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-207-6_3 Epithelial mesenchymal transition (EMT) is a multi-stage process whereby epithelial cells lose their cell:cell adhesions and acquire the capacity to migrate independently. It is a process that is important in normal development and is thought to be adopted by some invasive cancer cells. EMT requires modifications in cell shape and substratum adhesions and these events are dependent on the reorganisation of the actin cytoskeleton. Hepatocyte growth factor (HGF) is a mitogenic growth factor that is well known to induce such a conversion, termed “cell scattering”, in Madin Darby canine kidney (MDCK) cells. Recently, we have developed an alternative model of cell scattering using the human prostate cancer cell line, DU145. Like MDCK cells, DU145 cells normally grow as tight colonie... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5019061 Wed, 13 Jul 2011 01:02:39 +0100 5019061 http://www.medworm.com/index.php?rid=5019060&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-207-6_30 Cell migration is required for a wide variety of processes from bacteria seeking for food to correct patterning of neuronal networks. The ability to sense external cues is critical for cells to get directions and reach their goals. So far, studies on chemotaxis have mainly focused their attention on individual cells and therefore available tools are designed to monitor cell behavior at the single cell level. However, as collective cell migration is now widely accepted as a main mode of cell migration from development to cancer, the question of how chemotaxis is achieved has also to be asked on a bigger scale. Here, we present two chemotaxis assays suitable for single cells, cell sheets, and cell explants. Using a simple combination of heparin-coated beads and high vacuum silicone grease, t... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5019060 Wed, 13 Jul 2011 01:02:39 +0100 5019060 http://www.medworm.com/index.php?rid=5019059&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-207-6_2 The scratch-wound assay is a simple, reproducible assay commonly used to measure basic cell migration parameters such as speed, persistence, and polarity. Cells are grown to confluence and a thin “wound” introduced by scratching with a pipette tip. Cells at the wound edge polarise and migrate into the wound space. Advantages of this assay are that it does not require the use of specific chemoattractants or gradient chambers and it generates a strong directional migratory response, even in cell types that do not show robust responses in “single cell” migration assays. It is most reliably analysed when performed using time-lapse imaging, which can also yield valuable cell morphology/protein localisation information (Table 1). (Source: Springer protocols feed by Cell B... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5019059 Wed, 13 Jul 2011 01:02:39 +0100 5019059 http://www.medworm.com/index.php?rid=5019058&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-207-6_28 This chapter describes a method to study cells migrating in micro-channels, a confining environment of well-defined geometry. This assay is a complement to more complex 3D migration systems and provides several advantages even if it does not recapitulate the full complexity of 3D migration. Important parameters such as degree of adhesion, degree of confinement, mechanical properties, and geometry can be varied independently of each other. The device is fully compatible with almost any type of light microscopy and the simple geometry makes automated analysis very easy to perform, which allows screening strategy. The chapters is divided into five parts describing the design of different types of migration chambers, the fabrication of a mold by photolithography, the assembly of the chamber, t... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5019058 Wed, 13 Jul 2011 01:02:38 +0100 5019058 http://www.medworm.com/index.php?rid=5019057&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-207-6_27 Cell migration is a process that is controlled by the formation and correct localization of protein complexes and by post-translational modification of individual proteins. Forster or fluorescent resonance energy transfer (FRET) detected using fluorescence lifetime imaging microscopy (FLIM) provides a method by which protein–protein interactions may be detected and spatially localized within a cell. This technique can be used to map protein activation states and the formation and dissolution of protein complexes that control movement of a cell. This chapter describes a protocol for detecting FRET between GFP- and mRFP1-tagged proteins in fixed adherent cells. A background to both FRET and FLIM is provided followed by an overview of the method and a full protocol for sample preparatio... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5019057 Wed, 13 Jul 2011 01:02:38 +0100 5019057 http://www.medworm.com/index.php?rid=5019056&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-207-6_26 This chapter describes the use of microscope-based fluorescence recovery after photobleaching (FRAP). To quantify the dynamics of proteins within a subcellular compartment, we first outline the general aspects of FRAP experiments and then provide a detailed protocol of how to measure and analyse the most important parameters of FRAP experiments such as mobile fraction and half-time of recovery. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5019056 Wed, 13 Jul 2011 01:02:38 +0100 5019056 http://www.medworm.com/index.php?rid=5019055&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-207-6_25 Total internal reflection fluorescence microscopy (TIRF-M) has become an increasingly popular tool to study events in close proximity to the cell cortex, such as cell adhesion (Axelrod, J Cell Biol 89:141–145, 1981; Gingell et al., J Cell Biol 100:1334–1338, 1985; Patel et al., J Cell Sci 121:1159–1164, 2008), actin (Bretschneider et al., Curr Biol 14:1–10, 2004; Gerisch, Biophys J 87:3493–3503, 2004; Merrifield et al., Nat Cell Biol 4:691–698, 2002), and membrane dynamics (Oheim et al., Eur Biophys J 27:83–98, 1998; Steyer et al., Nature 388:474–478, 1997; Weisswange et al., J Cell Sci 118:4375–4380, 2005). In TIRF-M, dim fluorescence from cortical structures can be imaged with high contrast despite large cytoplasmic background from th... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5019055 Wed, 13 Jul 2011 01:02:38 +0100 5019055 http://www.medworm.com/index.php?rid=5019054&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-207-6_24 The chick embryo is easily accessible and has therefore been widely used in developmental biology studies. In particular, the early embryo can be removed from the egg and cultured, which allows real-time observations and imaging. Here, we describe ex vivo electroporation followed by long-term time-lapse microscopy, image capture, and processing. We have applied this approach to characterise the migration route of cardiac progenitor cells (CPCs) in live embryos. The heart is the first organ to function during vertebrate development and it is essential for the continued growth and survival of the embryo. In the chick, cardiac progenitors have been mapped to the anterior and mid-primitive streak at Hamburger–Hamilton stage 3. However, until recently it was not possible to observe cell m... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5019054 Wed, 13 Jul 2011 01:02:37 +0100 5019054 http://www.medworm.com/index.php?rid=5019053&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-207-6_23 Angiogenesis is the formation of new blood vessels from pre-existing vessels. This process is essential during embryonic development, wound healing, and regeneration as well as during several pathological conditions such as cancer. In this chapter, we describe an assay to measure angiogenesis in vivo in mice – the subcutaneous sponge assay. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5019053 Wed, 13 Jul 2011 01:02:37 +0100 5019053 http://www.medworm.com/index.php?rid=5019052&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-207-6_22 In the last decade, intravital microscopy on breast tumours in mice at single-cell resolution has resulted in important new insight into mechanisms of metastatic behaviour such as migration, invasion, and intravasation of tumour cells; angiogenesis; and the response of immune cells. This chapter describes the methods that can be used for analysing tumour cell motility in a mouse model of breast cancer metastasis. It includes protocols for generation of a labelled primary tumour, its imaging with two-photon microscopy, and the processing of time-lapse image data. Furthermore, we present a methodology, recently developed in our laboratory that combines multicolour imaging with an inducible cell model to study the role of a specific gene of interest in tumour cell motility in vivo. This proto... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5019052 Wed, 13 Jul 2011 01:02:37 +0100 5019052 http://www.medworm.com/index.php?rid=5019051&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-207-6_21 Many steps of the metastatic cascade can be reproduced in simple in vitro assays such as tumour cell interactions with matrix proteins, proteolysis, chemotaxis, haptotaxis, and invasion into matrices or explanted tissues. Nevertheless, there are no fully adequate substitutes for the complexity of the in vivo process. Here, we describe two “experimental” metastasis assays to yield lung or liver colonies (mimicking established micrometastatic disease), and two spontaneous metastasis assays for breast and prostate carcinomas. Examples include either murine tumour cell lines in syngeneic immunocompetent mice or human tumour xenografts in immunodeprived mice. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5019051 Wed, 13 Jul 2011 01:02:37 +0100 5019051 http://www.medworm.com/index.php?rid=5019050&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-207-6_1 Cell migration is a fundamental process that controls morphogenesis and inflammation. Its deregulation causes or is part of many diseases, including autoimmune syndromes, chronic inflammation, mental retardation, and cancer. Cell migration is an integral part of the cell biology, embryology, immunology, and neuroscience fields; as such, it has benefited from quantum leaps in molecular biology, biochemistry, and imaging techniques, and the emergence of the genomic and proteomic era. Combinations of these techniques have revealed new and exciting insights that explain how cells adhere and move, how the migration of multiple cells are coordinated and regulated, and how the cells interact with neighboring cells and/or react to changes in their microenvironment. This introduction provides a pri... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5019050 Wed, 13 Jul 2011 01:02:37 +0100 5019050 http://www.medworm.com/index.php?rid=5019049&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-207-6_19 Border cell migration in the Drosophila ovary has emerged as a genetically tractable model for studying collective cell movement. Over many years border cell migration was exclusively studied in fixed samples due to the inability to culture stage 9 egg chambers in vitro. Although culturing late stage egg chambers was long feasible, stage 9 egg chambers survived only briefly outside the female body. We identified culture conditions that support stage 9 egg chamber development and sustain complete migration of border cells ex vivo. This protocol enables one to compare the dynamics of egg chamber development in wild type and mutant egg chambers using time-lapse microscopy and taking advantage of a multiposition microscope with a motorized imaging stage. In addition, this protocol has been suc... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5019049 Wed, 13 Jul 2011 01:02:37 +0100 5019049 http://www.medworm.com/index.php?rid=5019048&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-207-6_18 A key feature of inflammatory cells is the ability to migrate to a site of injury or infection quickly and efficiently. Infectious agents can then be taken up by these inflammatory cells, preventing established infection. Inflammatory cell migration is driven by a complex interaction between inflammatory cells and their environment. In order to maintain health, inflammation needs to resolve, allowing the surrounding tissues to recover and heal. These processes are not fully understood and have been difficult to study in cell culture due to the complex interactions between cell types. We therefore use a range of techniques in near-transparent zebrafish (Danio rerio) larvae to study these migration events in a whole-organism, in vivo model. Using a transgenic zebrafish line that specifically... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5019048 Wed, 13 Jul 2011 01:02:37 +0100 5019048 http://www.medworm.com/index.php?rid=5019047&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-207-6_17 This protocol describes an in vivo assay for random and directed hemocyte migration in Drosophila. Drosophila is becoming an increasingly powerful model system for in vivo cell migration analysis, combining unique genetic tools with translucency of the embryo and pupa, which allows direct imaging and traceability of different cell types. In the assay we present here, we make use of the hemocyte response to epithelium wounding to experimentally induce a transition from random to directed migration. Time-lapse confocal microscopy of hemocyte migration in untreated conditions provides a random cell migration assay that allows identification of molecular mechanisms involved in this complex process. Upon laser-induced wounding of the thorax epithelium, a rapid chemotactic response changes hemoc... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5019047 Wed, 13 Jul 2011 01:02:36 +0100 5019047 http://www.medworm.com/index.php?rid=5019046&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-207-6_16 The nematode Caenorhabditis elegans is an excellent model system in which to study long-distance cell migration in vivo. This chapter describes methods used to study a subset of migratory cells in the hermaphrodite nematode, the distal tip cells. These methods take advantage of the organism’s transparent body and the expression of green fluorescent protein to observe cell migration and behavior. Additionally, the availability of nematode mutants and gene knockdown techniques that affect cell migration allow the analysis and comparison of wild-type and aberrant migratory paths. Methods for nematode growth and maintenance, strain acquisition, observation and live imaging, gene knockdown, and analysis of cell migration defects are covered. (Source: Springer protocols feed by Cell Biolog... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5019046 Wed, 13 Jul 2011 01:02:36 +0100 5019046 http://www.medworm.com/index.php?rid=5019045&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-207-6_15 Organotypic cultures are in vitro models that can be used to study the interactions between tumour and stromal cells. Collective tumour cell invasion in organotypic assays resembles that seen in human tissues in vivo, suggesting physiological relevance. A qualitative, pathological description of such invasion may be inadequate, and there is therefore a need to accurately quantify the degree of invasion. Although the simplest method to quantify invasion is to measure maximum invasive depth, this ignores the importance of the pattern of tumour invasion, which often reflects tumour aggressiveness. We use image analysis software to analyse organotypic invasion objectively, taking into account the average depth of tumour invasion, and the number and area of invading tumour islands. The product ... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5019045 Wed, 13 Jul 2011 01:02:36 +0100 5019045 http://www.medworm.com/index.php?rid=5019044&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-207-6_14 The use of fluorescent tags for in vivo tracking of proteins has provided an array of new data on cell function. Correspondingly, a variety of new methods utilizing these fluorescent tags have been developed. These methods must take into account all of the concerns of keeping live samples in conditions as close to physiological norms as possible, including temperature, CO2 levels, media composition, and reduction of phototoxic effects. The microscope itself should also be designed to maximize the benefits and minimize the risks inherent in these methods. We provide an overview of these concerns. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5019044 Wed, 13 Jul 2011 01:02:36 +0100 5019044 http://www.medworm.com/index.php?rid=5019043&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-207-6_13 We present a microfabricated chamber designed for visualising and quantifying the chemotaxis of slow-migrating adherent mammalian cells such as cancer and endothelial cells. Most of the existing solutions for the investigation of chemotaxis are limited to fast migrating cells such as leukocytes or Dictyostelium discoideum. Here, we describe the details of an assay using the μ-Slide Chemotaxis to investigate the chemotactic response of human umbilical vein endothelial cells to a gradient of human vascular endothelial growth factor 165. In combination with phase contrast video microscopy and cell tracking, the trajectories of all single cells migrating in temporally stable gradients are derived. The resulting migration data are displayed and analysed in detail by several different paramet... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5019043 Wed, 13 Jul 2011 01:02:36 +0100 5019043 http://www.medworm.com/index.php?rid=5019042&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-207-6_12 Migration of cells across endothelial barriers, termed transendothelial migration (TEM), is an important cellular process that underpins the pathology of many disease states including chronic inflammation and cancer metastasis. While this process can be modeled in vitro using cultured cells, many model systems are unable to provide detailed visual information of cell morphologies and distribution of proteins such as junctional markers, as well as quantitative data on the rate of TEM. Improvements in imaging techniques have made microscopy-based assays an invaluable tool for studying this type of detailed cell movement in physiological processes. In this chapter, we describe a confocal microscopy-based method that can be used to assess TEM of both leukocytes and cancer cells across endothel... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5019042 Wed, 13 Jul 2011 01:02:36 +0100 5019042 http://www.medworm.com/index.php?rid=5019041&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-207-6_11 Cell migration on two-dimensional (2D) substrates follows entirely different rules than cell migration in three-dimensional (3D) environments. This is especially relevant for leukocytes that are able to migrate in the absence of adhesion receptors within the confined geometry of artificial 3D extracellular matrix scaffolds and within the interstitial space in vivo. Here, we describe in detail a simple and economical protocol to visualize dendritic cell migration in 3D collagen scaffolds along chemotactic gradients. This method can be adapted to other cell types and may serve as a physiologically relevant paradigm for the directed locomotion of most amoeboid cells. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5019041 Wed, 13 Jul 2011 01:02:36 +0100 5019041 http://www.medworm.com/index.php?rid=5019040&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-207-6_10 The endothelial cells lining blood vessels are continuously exposed to fluid shear stress generated by pulsatile flow of blood. In order to minimise forces acting on their surface, endothelial cells adapt to shear stress by alignment and migration within the direction of flow. Failure to adapt to shear stress results in endothelial damage contributing to generation of atherosclerotic plaques or abnormal vessel repair. This chapter describes methods of generating laminar flow in vitro and studying endothelial cell alignment and motility under flow. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=5019040 Wed, 13 Jul 2011 01:02:36 +0100 5019040 http://www.medworm.com/index.php?rid=4984219&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-185-7_4 Tight junctions (TJs) are intercellular structures in epithelial and endothelial cells, primarily playing ­critical roles in cell–cell adhesion. Among their molecular components, claudins are the main constituents as integral membrane proteins, encoded by at least 24 members of a single gene family. Accumulated ­evidence has demonstrated that TJ proteins such as claudins are directly involved in the regulation of ­cellular functions such as proliferation, differentiation, and apoptosis, due to their ability to recruit various signaling molecules that have proliferative and differentiative capacities, including transcription factors, lipid phosphatases, and cell cycle regulators. It is thus clear that TJs are not simple static constituents to establish cell adhesion struct... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=4984219 Thu, 30 Jun 2011 16:27:58 +0100 4984219 http://www.medworm.com/index.php?rid=4984218&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-185-7_3 Claudins are tight junction membrane proteins that act as paracellular pores and barriers and regulate epithelial permeability to small ions. A key step in understanding the function of any claudin isoform is the in vitro measurement of its ion permeability and selectivity. Herein, we describe methods to generate clonal lines with stable inducible overexpression of claudins in Madin–Darby canine kidney epithelial cells, measure conductance and diffusion potentials in Ussing chambers, correct for liquid junction potentials, and derive quantitatively accurate values for individual ion permeabilities. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=4984218 Thu, 30 Jun 2011 16:27:58 +0100 4984218 http://www.medworm.com/index.php?rid=4984217&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-185-7_30 Anti-claudin-4, whose corresponding antigen receptors are known to be overexpressed in both primary and metastatic human pancreatic cancer, is utilized for targeted delivery and imaging of pancreatic cancer. In this protocol, we describe the use of quantum dots (QDs) as sensitive optical contrast agent for imaging pancreatic cancer in vitro and in vivo by using anti-claudin-4 as targeting ligands. The claudin-4-mediated targeting is demonstrated in using both in vitro confocal microscopy and in vivo tumor imaging system. This targeted QD platform will be further modified for the purpose of developing as an early detection imaging tool for pancreatic cancer. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=4984217 Thu, 30 Jun 2011 16:27:58 +0100 4984217 http://www.medworm.com/index.php?rid=4984216&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-185-7_2 Tissue barrier function is directly mediated by tight junction transmembrane proteins known as claudins. Cells that form tight junctions typically express multiple claudin isoforms, which suggests that heterotypic (head-to-head) binding between different claudin isoforms may play a role in regulating paracellular permeability. To test whether claudins are heterotypically compatible, we developed an assay system using HeLa cells, a claudin-null cell line which expresses other tight junction proteins, including occludin, junction adhesion molecule A, and zonula occludens-1, -2, and -3. HeLa cells stably transfected to express different claudins are cocultured, then subsequently analyzed for the ability to coimmunopurify. Using this approach, we have found that claudin-1, claudin-3, and claud... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=4984216 Thu, 30 Jun 2011 16:27:58 +0100 4984216 http://www.medworm.com/index.php?rid=4984215&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-185-7_29 Claudins comprise a multigene family of 24 species and have been shown to constitute the backbone of tight junction strands in simple epithelial cells and to be directly involved in their barrier functions. Apical-most tight junction protein complexes (TJs) are implicated in inflammatory bowel disease (IBD) pathophysiology. Except for claudin-8, TJs explored in this study (including ZO-1, claudin-1, -2, -3, -7, -12, and -15) were found to be expressed in rat colonic tissues. ZO-1 and claudin-7 were ubiquitously expressed in all study groups. As depicted in Fig. 1b, expressions of claudin-2, -12, and -15 significantly diminished after combined treatment with dextran sulfate sodium (DSS) and busulfan (BU) (lane 5), compared with either agent alone (lanes 2 and 4). Despite the lack of signifi... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=4984215 Thu, 30 Jun 2011 16:27:58 +0100 4984215 http://www.medworm.com/index.php?rid=4984214&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-185-7_28 We examined expression of claudin-16 in human breast cells and tissues to identify a possible link between expression and aggressiveness in cells and between claudin-16 levels and patient prognosis. Forced expression of claudin-16 in breast cancer cells resulted in a less aggressive phenotype and reduced in vivo tumour ­volume. Claudin-16 expression was reduced in human breast cancer, particularly in patients with aggressive tumours and high mortality (Martin et al. J Cell Biochem 105:41–52,2008). This suggests that claudin-16 plays a role beyond that of an initial metastasis repressor in this cancer type. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=4984214 Thu, 30 Jun 2011 16:27:57 +0100 4984214 http://www.medworm.com/index.php?rid=4984213&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-185-7_27 There are a number of disadvantages with conventional tissue immunohistochemistry for accurate ­localisation of claudin proteins. Traditionally, tissue cryopreservation or formaldehyde fixation with wax embedding is utilised prior to sectioning and antibody localisation. Wax embedding gives better morphological preservation than frozen tissue, but the required use of chemical cross-linking fixatives renders many antigens inaccessible to antibody binding or results in subsequent disruption of antibody localisation patterns due to the use of harsh antigen retrieval methods. Use of frozen or wax-embedded tissue also requires the cutting of relatively thick >6-μm sections, making the interrogation of serial sections very limited. The use of glycolmethacrylate (GMA) tissue embedding w... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=4984213 Thu, 30 Jun 2011 16:27:57 +0100 4984213 http://www.medworm.com/index.php?rid=4984212&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-185-7_26 HIV-1 crosses the blood–brain barrier (BBB) early in the course of systemic infection and resides in brain macrophages and microglia. The integrity of the brain endothelium is regulated by intercellular tight junctions, which also play a critical role in HIV-1-entry into the brain. Disruption of tight junctions, including changes in claudin-5 expression, is common in HIV-1-infected patients. Recent evidence indicates that both exposure to HIV-1 and HIV-1 specific proteins, such as Tat protein, can contribute to alterations of expression and distribution of claudin-5 in brain endothelial cells and brain microvessels. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=4984212 Thu, 30 Jun 2011 16:27:57 +0100 4984212 http://www.medworm.com/index.php?rid=4984211&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-185-7_25 Claudins are transmembrane proteins that form the backbone of the tight junctions (TJs) at the blood–brain barrier (BBB). TJs are cellular structures that physically obstruct the inter-endothelial space and restrict the paracellular diffusion of blood-borne substances from the peripheral circulation into the CNS. TJs are also dynamic structures that rapidly respond to external signals that produce changes in BBB permeability. We focus here on the biochemical and immunohistochemical properties of claudin-5 as expressed in three in vitro models of the BBB, and show that the contact co-culture of endothelial cells with glial cells significantly increases claudin-5 expression. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=4984211 Thu, 30 Jun 2011 16:27:57 +0100 4984211 http://www.medworm.com/index.php?rid=4984210&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-185-7_24 The blood–brain barrier (BBB) has become a major focus of attention in cerebral pathophysiology and disease progression in the central nervous system. Endothelial tight junctions, the basal lamina, and perivascular astrocytes are jointly referred to as BBB or neurovascular unit. Around the cerebral endothelial cells is the basal lamina composed primarily of laminin, fibronectin, and heparan sulfate. The basal lamina provides a structural barrier to extravasation of cellular blood elements and anchors endothelial cells to astrocytes. Barriers limiting transport into and out of the brain are found at the tight junction proteins and at the basal lamina. The relative contribution of these two sites has not been studied, but it is likely that both are disrupted to some extent in various i... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=4984210 Thu, 30 Jun 2011 16:27:57 +0100 4984210 http://www.medworm.com/index.php?rid=4984209&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-185-7_23 It is apparent that claudins are involved in signalling to and from cellular tight junctions (TJs) and control cell behaviour such as proliferation, differentiation, and migration. Methods to identify and measure specific claudins in TJs would, therefore, be useful to monitor TJ structure and functional integrity under physiological and pathological conditions. The molecular pathways involved in claudin signalling are not understood and are likely to become a focus for intensive research as better understanding of tight junction structure and function may provide opportunities for better drug delivery and absorption. In this chapter, we describe our method for quantitative analysis of specific claudins in TJ during the breakdown of the blood–retinal barrier in a mouse model of inflam... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=4984209 Thu, 30 Jun 2011 16:27:57 +0100 4984209 http://www.medworm.com/index.php?rid=4984208&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-185-7_22 Claudin-5 is a transmembrane tight junction protein highly expressed in brain endothelial cells, the site of the blood–brain barrier. The properties of the brain endothelial tight junction complex are considered to be dependent on claudin-5 cell–cell interaction, putting this protein in a position to play a major role in the maintenance of brain endothelial barrier integrity. Thus, alterations in claudin-5 function can lead to “opening” of the paracellular route and increased brain endothelial barrier permeability. Recent work from the authors’s laboratory has established that caveolae-dependent internalization/recycling of claudin-5 is a mechanism underlying transient increases in brain endothelial paracellular permeability in the presence of pro-inflammatory... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=4984208 Thu, 30 Jun 2011 16:27:57 +0100 4984208 http://www.medworm.com/index.php?rid=4984207&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-185-7_21 Claudins are the most important components of the tight junctions at the interface of the basolateral and apical membranes of polarized epithelial and endothelial cells. They determine the barrier properties of cell–cell contact existing between two neighboring cells and regulate paracellular permeability. Although maintenance of barrier properties requires intact epithelial tight junctions, relatively little is known about the role of claudins expressed in the alveolar epithelium in the regulation of epithelial permeability in response to inflammatory stimuli and oxidative stress, or injury. The present study was undertaken to determine whether differential expression of tight junction claudins is a mechanism for regulation of oxidant-induced pulmonary epithelial hyperpermeability. ... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=4984207 Thu, 30 Jun 2011 16:27:57 +0100 4984207 http://www.medworm.com/index.php?rid=4984206&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-185-7_20 Immunohistochemistry is a suitable method for the detection of proteins from the Claudin family and several antibodies are commercially available for the detection of Claudin congeners. Immunodetection of Caludin-4 in the paraffin-embedded specimens might be a useful tool for studying the role of these proteins in the cyclic transformation of the endometrium and its role in the endometrial receptivity; furthermore, other components of the junctional zone involved in the transformational process of the endometrium can be detected by means of immunohistochemistry/immunofluorescence with several polyclonal or monoclonal antibodies. The aim of this chapter is to comprehensively overview the materials and methods to perform the endometrial biopsy and to detect Claudin-4 in paraffin-embedded sam... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=4984206 Thu, 30 Jun 2011 16:27:57 +0100 4984206 http://www.medworm.com/index.php?rid=4984205&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-185-7_1 Tight junctions restrict the paracellular movement of ions, solutes, drugs, and larger material across epithelia and endothelia. For practical purposes, the barrier can be modeled as having two components. The first is a system of small 4  Å radius pores lined or created by claudins. The pores show variable ionic charge selectivity and electrical resistance based on the pattern of claudin proteins expressed in a particular junction. Transport of compounds that are larger than 4  Å are not subject to discrimination based on size or charge; they are likely passing through transient breaks in the tight junction barrier. The magnitude of the first and second pathways varies among epithelia and is altered in response to physiological and pathological stimuli. Unfortuna... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=4984205 Thu, 30 Jun 2011 16:27:56 +0100 4984205 http://www.medworm.com/index.php?rid=4984204&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-185-7_19 The examination of the epithelial barrier has been a primary site of focused research for years. Despite the importance of this site to numerous intestinal diseases, the determination of the integrity of this barrier has been clouded by controversies as to the validity of certain techniques and the ease of use regarding others. To determine the barrier integrity in vivo, we have adapted a simple tracer-based microscopic assay that was initially used in other systems to the in vivo intestinal epithelium. This technique is widely adaptable to other tracer molecules that can be applied to the tissue and consequently generates images depicting barrier maintenance or functional breach. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=4984204 Thu, 30 Jun 2011 16:27:56 +0100 4984204 http://www.medworm.com/index.php?rid=4984203&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-185-7_18 It is estimated that between 12 and 15% of couples are infertile. More than half of these are related to problems associated with male reproductive dysfunction. Of those, 40% occur from idiopathic or unexplained causes. While spermatozoa are formed in the testis, testicular spermatozoa are immature and cannot swim or fertilize. These critical functions are acquired as spermatozoa transit through the epididymis in the specific luminal environment created in part by the tight junctions of the blood-epididymis barrier. To understand the normal and pathological conditions attributable to human and animal epididymal function, we have needed to develop biological tools to characterize the physiological, cellular, and molecular functions of tight junctions and claudins (Cldns) in the epididymis. ... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=4984203 Thu, 30 Jun 2011 16:27:56 +0100 4984203 http://www.medworm.com/index.php?rid=4984202&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-185-7_17 Primary saliva is produced from blood plasma in the acini of salivary glands and is modified by ion adsorption and secretion as the saliva passes through the ducts. In rodents, acinar cells of salivary glands express claudin-3 but not claudin-4, whereas duct cells express both claudins-3 and -4. The distinct claudin expression patterns may reflect differences in the permeability of tight junctions between acinar and duct cells. To analyze the role of claudins in salivary glands, we established a system for the primary culture of parotid acinar cells, where the expression patterns of claudins are remarkably changed. Real-time RT–PCR and immunoblot analyses reveal that the expression levels of claudins-4 and -6 increased, whereas claudins-3 and -10 decreased. We found that the signal t... Springer protocols feed by Cell Biology news http://www.medworm.com/rss/comments.php?id=4984202 Thu, 30 Jun 2011 16:27:56 +0100 4984202