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 'Current Protocols in Cell Biology' source. Sat, 20 Mar 2010 16:35:11 +0100 http://www.medworm.com/index.php?rid=3382167&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20235099%26dopt%3DAbstract Authors: Hodgson L, Shen F, Hahn K The biosensors developed in the authors' laboratory have been based on different designs, each imparting specific strengths and weaknesses. Here we describe detailed protocols for the application of three biosensors exemplifying different designs-first, a design in which an environmentally sensitive dye is used to report the activation of endogenous Cdc42, followed by two biosensors based on FRET, one using intramolecular and the other intermolecular FRET. The design differences lead to the need for different approaches in imaging and image analysis. Curr. Protoc. Cell Biol. 46:14.11.1-14.11.26. (c) 2010 by John Wiley & Sons, Inc. PMID: 20235099 [PubMed - in process] (Source: Current Protocols in Cell Biology) Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=3382167 Mon, 01 Mar 2010 00:00:00 +0100 3382167 http://www.medworm.com/index.php?rid=3382166&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20235100%26dopt%3DAbstract Authors: Sorkin A, Duex JE Binding of epidermal growth factor (EGF) to the EGF receptor (EGFR) initiates signal transduction, ultimately leading to altered gene expression. Ligand-activated EGFR is also rapidly internalized and then targeted to lysosomes for degradation or recycled back to the plasma membrane. Endocytosis is a major regulator of EGFR signaling. Therefore, elucidation of the mechanisms of EGFR endocytosis is essential for a better understanding of EGFR biology. In order to achieve a comprehensive analysis of these mechanisms, reliable methods for measuring the rates of EGFR protein turnover and the rate parameters for individual steps of EGFR endocytic trafficking must be employed. The protocols in this unit describe methodologies to measure the rates of EGFR synthesis ... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=3382166 Mon, 01 Mar 2010 00:00:00 +0100 3382166 http://www.medworm.com/index.php?rid=3382165&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20235101%26dopt%3DAbstract Authors: Ishikura S, Antonescu CN, Klip A The elevated blood glucose following a meal is cleared by insulin-stimulated glucose entry into muscle and fat cells. The hormone increases the amount of the glucose transporter GLUT4 at the plasma membrane in these tissues at the expense of preformed intracellular pools. In addition, muscle contraction also increases glucose uptake via a gain in GLUT4 at the plasma membrane. Regulation of GLUT4 levels at the cell surface could arise from alterations in the rate of its exocytosis, endocytosis, or both. Hence, methods that can independently measure these traffic parameters for GLUT4 are essential to understanding the mechanism of regulation of membrane traffic of the transporter. Here, we describe cell population-based assays to measure the stea... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=3382165 Mon, 01 Mar 2010 00:00:00 +0100 3382165 http://www.medworm.com/index.php?rid=3382164&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20235102%26dopt%3DAbstract Authors: Boukhvalova MS, Yim KC, Prince GA, Blanco JC Viral infection is normally detected either by viral culture or by PCR methods. Rarely is a combination of the two techniques used in the same study. Yet, when applied simultaneously, viral culture and PCR may reveal important features of viral biology, such as an abortive replication, as in the case of respiratory syncytial virus (RSV) infection. In this unit, we describe methods for detecting abortive RSV replication in a cotton rat model by using the plaque-forming unit assay and the real-time reverse-transcription PCR (qRT-PCR) assay. All steps of the process of monitoring viral replication in vivo are described, starting from the design of animal infection protocols. We continue on to the methods for extracting and processing l... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=3382164 Mon, 01 Mar 2010 00:00:00 +0100 3382164 http://www.medworm.com/index.php?rid=3382163&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20235103%26dopt%3DAbstract Authors: Urwyler S, Finsel I, Ragaz C, Hilbi H The environmental bacterium Legionella pneumophila naturally parasitizes free-living amoebae. L. pneumophila is an opportunistic human pathogen that grows in macrophages, thus causing a life-threatening pneumonia termed Legionnaires' disease. The bacteria replicate intracellularly in environmental and immune phagocytes within a unique compartment, the Legionella-containing vacuole (LCV). Formation of LCVs is a complex and robust process involving >150 secreted bacterial effector proteins, which are believed to subvert host cell signaling and vesicle trafficking pathways. This unit describes a simple approach to purify intact LCVs from Dictyostelium discoideum amoebae. The method comprises a two-step purification protocol that includes i... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=3382163 Mon, 01 Mar 2010 00:00:00 +0100 3382163 http://www.medworm.com/index.php?rid=3382162&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20235104%26dopt%3DAbstract Authors: Nießen J, Jedlitschky G, Greinacher A, Kroemer HK Functional analysis of platelet intracellular structures requires isolation and purification of these cellular compartments. With regard to the function of platelets, both, dense (delta) and alpha granules are relevant target structures. However, the availability of sufficient purification protocols for these structures is rather limited. This unit describes two protocols for isolation and purification of platelet granule structures. The Basic Protocol describes a new technique based on immunolabeling with target-specific antibodies followed by magnetic sorting, whereas the Alternate Protocol describes the more traditional procedure based on differential centrifugation and density-based sedimentation. For both methods, the... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=3382162 Mon, 01 Mar 2010 00:00:00 +0100 3382162 http://www.medworm.com/index.php?rid=3382146&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D20235105%26dopt%3DAbstract In conclusion, the protocols described here will aid the understanding of mitochondrial structure-function relationship. Curr. Protoc. Cell Biol. 46:4.25.1-4.25.21. (c) 2010 by John Wiley & Sons, Inc. PMID: 20235105 [PubMed - in process] (Source: Current Protocols in Cell Biology) Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=3382146 Mon, 01 Mar 2010 00:00:00 +0100 3382146 http://www.medworm.com/index.php?rid=2768418&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D19731224%26dopt%3DAbstract Authors: Hall B, Limaye A, Kulkarni AB The technique of gene targeting allows for the introduction of engineered genetic mutations into a mouse at a determined genomic locus. The process of generating mouse models with targeted mutations was developed through both the discovery of homologous recombination and the isolation of murine embryonic stem cells (ES cells). Homologous recombination is a DNA repair mechanism that is employed in gene targeting to insert a designed mutation into the homologous genetic locus. Targeted homologous recombination can be performed in murine ES cells through electroporation of a targeting construct. These ES cells are totipotent and, when injected into a mouse blastocyst, they can differentiate into all cell types of a chimeric mouse. A chimeric mouse ha... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=2768418 Mon, 31 Aug 2009 23:00:00 +0100 2768418 http://www.medworm.com/index.php?rid=2768417&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D19731225%26dopt%3DAbstract Authors: Limaye A, Hall B, Kulkarni AB The establishment of mouse embryonic stem (ES) cell lines has allowed for the gene?ration of the knockout mouse. ES cells that are genetically altered in culture can then be manipulated to derive a whole mouse containing the desired mutation. To successfully generate a knockout mouse, however, the ES cells must be carefully cultivated in a pluripotent state throughout the gene-targeting experiment. This unit describes detailed step-by-step protocols, reagents, equipment, and strategies needed for the successful generation of gene knockout embryonic stem cells using homologous recombination technologies. PMID: 19731225 [PubMed - in process] (Source: Current Protocols in Cell Biology) Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=2768417 Mon, 31 Aug 2009 23:00:00 +0100 2768417 http://www.medworm.com/index.php?rid=2768416&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D19731226%26dopt%3DAbstract Authors: Longenecker G, Kulkarni AB This unit describes protocols used in the production of chimeric mice that are then used for the generation of gene knockout mice. These protocols include the collection of blastocyst embryos, ES cell injection, and uterine transfer of injected blastocysts. Support protocols for the superovulation of blastocyst donor mice, generation of pseudopregnant recipients, fabrication of glass pipets for embryo and cell manipulations, and generation of germline mice are also included. Practical tips and solutions are mentioned to help troubleshoot problems that may occur. PMID: 19731226 [PubMed - in process] (Source: Current Protocols in Cell Biology) Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=2768416 Mon, 31 Aug 2009 23:00:00 +0100 2768416 http://www.medworm.com/index.php?rid=2768415&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D19731227%26dopt%3DAbstract Authors: Rostagno A, Ghiso J Extracellular deposits of amyloid fibrils in the form of parenchymal plaques and cerebrovascular lesions, as well as intracellular accumulation of paired-helical filaments in the form of neurofibrillary tangles (NFT) in selected neuronal populations are the main neuropathologic hallmarks of Alzheimer's disease. Amyloid fibrils composed of polymeric structures of the amyloid-beta (Abeta) concentrate at the center of senile plaques and accumulate in the walls of cerebral blood vessels, exhibiting extensive Congo red/thioflavin S staining. Intraneuronal NFT are composed of building blocks of aberrantly hyperphosphorylated species of the microtubule-associated protein tau, which accumulate in the perinuclear cytoplasm of vulnerable neurons in the form of paired... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=2768415 Mon, 31 Aug 2009 23:00:00 +0100 2768415 http://www.medworm.com/index.php?rid=2526143&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D19499504%26dopt%3DAbstract Authors: Eberth A, Ahmadian MR Small GTPases act as tightly regulated molecular switches governing a large variety of critical cellular functions. Their activity is controlled by two different biochemical reactions, GDP/GTP exchange and GTP hydrolysis. These very slow reactions require catalysis in cells by two kinds of regulatory proteins. While the guanine nucleotide exchange factors (GEFs) activate small GTPases by stimulating the slow exchange of bound GDP for the cellularly abundant GTP, GTPase-activating proteins (GAPs) accelerate the slow intrinsic rate of GTP hydrolysis by several orders of magnitude, leading to inactivation. There are a number of methods that can be used to characterize the specificity and activity of such regulators, to understand the effect of binding on the... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=2526143 Sun, 31 May 2009 23:00:00 +0100 2526143 http://www.medworm.com/index.php?rid=2526142&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D19499505%26dopt%3DAbstract Authors: Flaño E, Jewell NA, Durbin RK, Durbin JE This unit describes protocols for infecting the mouse respiratory tract, and assaying virus replication and host response in the lung. Respiratory infections are the leading cause of acute illness worldwide, affecting mostly infants and children in developing countries. The purpose of this unit is to provide a basic strategy and protocols to study the pathogenesis and immunology of respiratory virus infection using the mouse as an animal model. The procedures include: (1) basic techniques for mouse infection, tissue sampling, and preservation, (2) determination of viral titers, isolation and analysis of lymphocytes and dendritic cells using flow-cytometry, and (3) lung histology, immunohistochemistry, and in situ hybridization. ... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=2526142 Sun, 31 May 2009 23:00:00 +0100 2526142 http://www.medworm.com/index.php?rid=2526141&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D19499506%26dopt%3DAbstract We describe the assembly and use of compartmented neuronal cultures for in vitro study of directional infection of neurons by alpha herpesviruses. Selective application of viral inoculum to only one compartment ensures that the remainder of the neuron is not contaminated by input inoculum. This allows for quantification of viral spread, and unambiguous interpretation of immunofluorescence and electron microscopy images. PMID: 19499506 [PubMed - in process] (Source: Current Protocols in Cell Biology) Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=2526141 Sun, 31 May 2009 23:00:00 +0100 2526141 http://www.medworm.com/index.php?rid=2526140&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D19499507%26dopt%3DAbstract Authors: Janas AM, Wu L Characterization of HIV-1 interactions with host cells is critical for cell biology studies of HIV-1. This unit describes a set of methods and protocols to perform quantitative assays of HIV-1 binding, internalization, infection, and cell-cell transmission. The protocols include: (1) generating infectious single-cycle or replication-competent HIV-1 stocks, (2) an HIV-1 binding and internalization assay, (3) HIV-1 infection of target cells and quantification of viral infection, and (4) HIV-1 cell-cell transmission assays. These functional assays provide useful tools to quantitatively study HIV-1 infection and viral transmission. PMID: 19499507 [PubMed - in process] (Source: Current Protocols in Cell Biology) Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=2526140 Sun, 31 May 2009 23:00:00 +0100 2526140 http://www.medworm.com/index.php?rid=2526139&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D19499508%26dopt%3DAbstract Authors: Elia N, Lippincott-Schwartz J Epithelial cells grown in three-dimensional (3-D) cultures of extracellular matrix differentiate into a multicellular structure of polarized cells. This process shares many characteristics with the physiological development of an epithelial tissue and the formation of polarity in epithelial cells. Imaging 3-D cultures of polarized epithelial cells is therefore a powerful tool to study epithelial architecture and morphogenesis under close-to-physiological conditions. The new generation of confocal microscopes allows live-cell imaging of fluorescently tagged molecules in these cultures. This opens up new opportunities for studying how molecules behave and are distinguished asymmetrically within a 3-D setting. This unit discusses technical aspects fo... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=2526139 Sun, 31 May 2009 23:00:00 +0100 2526139 http://www.medworm.com/index.php?rid=2526138&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D19499509%26dopt%3DAbstract Authors: Kucik DF This unit describes the analysis of dynamic cell adhesion using a flow chamber assay. The flow chamber enables the researcher to reconstruct cell systems in the presence of shear stress to assay adhesion under well&defined forces. These assays are most commonly used to study leukocyte adhesion, either to cultured endothelial cell monolayers or to purified substrates, simulating physiological interactions of leukocytes with endothelial cells. This assay can be also be used to characterize transient adhesive events or adhesion strengthening even for cells that do not normally experience shear stress, because contact time between cells and substrates and anti&adhesive forces can be closely regulated by stopping and starting the flow. Flow chamber assays are also ... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=2526138 Sun, 31 May 2009 23:00:00 +0100 2526138 http://www.medworm.com/index.php?rid=2261223&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D19283728%26dopt%3DAbstract Authors: Haruyama N, Cho A, Kulkarni AB Cell biology research encompasses everything from single cells to whole animals. Recent discoveries concerning particular gene functions can be applied to the whole animal for understanding genotype-phenotype relationships underlying disease mechanisms. For this reason, genetically manipulated mouse models are now considered essential to correctly understand disease processes in whole animals. This unit reviews the basic mouse technologies used to generate conventional transgenic mice, which represent a gain-of-function approach. First, an overview of transgenic construct design is presented. This unit then explains basic strategies for the identification and establishment of independent transgenic mouse lines, followed by comments on historical ... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=2261223 Sun, 01 Mar 2009 05:00:00 +0100 2261223 http://www.medworm.com/index.php?rid=2261222&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D19283729%26dopt%3DAbstract Authors: Cho A, Haruyama N, Kulkarni AB This unit describes detailed step-by-step protocols, reagents, and equipment required for successful generation of transgenic mice using pronuclear injection. The experimental methods and practical tips given here will help guide beginners in understanding what is required and what to avoid in these standard protocols for efficiently generating transgenic mice. PMID: 19283729 [PubMed - in process] (Source: Current Protocols in Cell Biology) Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=2261222 Sun, 01 Mar 2009 05:00:00 +0100 2261222 http://www.medworm.com/index.php?rid=2261221&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D19283730%26dopt%3DAbstract Authors: Balla T, Várnai P This unit describes the method of following phosphoinositide dynamics in live cells. Inositol phospholipids have emerged as universal signaling molecules present in virtually every membrane of eukaryotic cells. Phosphoinositides are present in only tiny amounts as compared to structural lipids, but they are metabolically very active as they are produced and degraded by the numerous inositide kinase and phosphatase enzymes. Phosphoinositides control the membrane recruitment and activity of many membrane protein signaling complexes in specific membrane compartments, and they have been implicated in the regulation of a variety of signaling and trafficking pathways. It has been a challenge to develop methods that allow detection of phosphoinositides at the s... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=2261221 Sun, 01 Mar 2009 05:00:00 +0100 2261221 http://www.medworm.com/index.php?rid=2261220&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D19283731%26dopt%3DAbstract Authors: Li WJ, Tuan RS Nanofibers fabricated by electrospinning are morphological mimics of fibrous components of the native extracellular matrix, making nanofibrous scaffolds ideal for three-dimensional cell culture and tissue engineering applications. Although electrospinning is not a conventional technique in cell biology, the experimental setup may be constructed in a relatively straightforward manner, and the procedure can be carried out by individuals with limited engineering experience. Here, we detail a protocol for electrospinning of nanofibers and provide relevant specific details concerning the optimization of fiber formation (Basic Protocol 1). The protocol also includes conditions required for preparing biodegradable polymer solutions for the fabrication of nonwoven and a... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=2261220 Sun, 01 Mar 2009 05:00:00 +0100 2261220 http://www.medworm.com/index.php?rid=2261219&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D19283732%26dopt%3DAbstract Authors: Moriyama T, Sorokin A BK virus (BKV) can cause BKV nephritis in renal transplant patients and has become a significant reason for graft loss in this decade. BKV is latent in the urogenital tract and most likely is transported with the donor kidney to recipients. BKV replication occurs in the nucleus of human renal proximal tubular cells (HRPTEC) and daughter viruses are delivered to other cells to spread infection. A few in vitro studies have been reported about the mechanism and kinetics of BKV infection. However, there are still a lot of unknown factors regarding BKV infection. This unit describes the handling of BKV, BKV propagation, determination of titer and ability to infect cells, as well as purification and labeling of BKV in order to analyze BKV cell entry. PMID: ... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=2261219 Sun, 01 Mar 2009 05:00:00 +0100 2261219 http://www.medworm.com/index.php?rid=2261218&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D19283733%26dopt%3DAbstract Authors: Hutton JC, Wong R, Davidson HW Methods are presented for the separation of dense core secretory vesicles from insulin-secreting tissues (insulin granules) based on a combination of differential and density gradient centrifugation on various media. Emphasis is given to the use of transplantable tumors, tissue culture cell lines, and pancreatic islets as a tissue source. PMID: 19283733 [PubMed - in process] (Source: Current Protocols in Cell Biology) Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=2261218 Sun, 01 Mar 2009 05:00:00 +0100 2261218 http://www.medworm.com/index.php?rid=2042818&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D19085985%26dopt%3DAbstract Authors: Sakai T, Onodera T This unit provides detailed protocols for dissecting embryonic organs and performing organ culture to study questions in developmental biology. Procedures are described here for dissecting organs such as kidney, lung, and salivary gland. The unit also contains commentary including troubleshooting for embryonic organ culture. Curr. Protoc. Cell Biol. 41:19.8.1-19.8.8. (c) 2008 by John Wiley & Sons, Inc. PMID: 19085985 [PubMed - in process] (Source: Current Protocols in Cell Biology) Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=2042818 Mon, 01 Dec 2008 05:00:00 +0100 2042818 http://www.medworm.com/index.php?rid=2042817&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D19085986%26dopt%3DAbstract Authors: Carlson MW, Alt-Holland A, Egles C, Garlick JA Over the last decade, the development of in vitro, human, three-dimensional (3D) tissue models, known as human skin equivalents (HSEs), has furthered understanding of epidermal cell biology and provided novel experimental systems. Signaling pathways that mediate the linkage between growth and differentiation function optimally when cells are spatially organized to display the architectural features seen in vivo, but are uncoupled and lost in two-dimensional culture systems. HSEs consist of a stratified squamous epithelium grown at an air-liquid interface on a collagen matrix populated with dermal fibroblasts. These 3D tissues demonstrate in vivo-like epithelial differentiation and morphology, and rates of cell division, similar to... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=2042817 Mon, 01 Dec 2008 05:00:00 +0100 2042817 http://www.medworm.com/index.php?rid=2042816&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D19085987%26dopt%3DAbstract Authors: Tokunaga K, Tani T Transport of mRNA from the nucleus to the cytoplasm is an essential process for gene expression in eukaryotic cells. In this unit, methods for monitoring nuclear mRNA export are described. Visualization of cellular mRNAs by fluorescence in situ hybridization with oligo(dT) probes is effectively applied to monitoring mRNA export from the nucleus in yeast and mammalian cells. In addition to the protocols for fluorescence in situ hybridization, this unit includes an alternate method that the authors have been developing for visual analysis of nuclear mRNA export in living mammalian cells by microinjection of fluorescently labeled pre-mRNA into the nuclei. Curr. Protoc. Cell Biol. 41:22.13.1-22.13.20. (c) 2008 by John Wiley & Sons, Inc. PMID: 19085987 [P... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=2042816 Mon, 01 Dec 2008 05:00:00 +0100 2042816 http://www.medworm.com/index.php?rid=2042815&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D19085988%26dopt%3DAbstract Authors: Tribl F Neuromelanin granules are pigmented organelles in the human midbrain that give name to a brain area, substantia nigra pars compacta, which macroscopically appears as a dark brown region in the midbrain due to the insoluble pigment neuromelanin. The substantia nigra pars compacta massively degenerates in Parkinson's disease and gives rise to severely disabling movement symptoms. It has been suggested that neuromelanin granules play an important role in the neurodegenerative events in Parkinson's disease: redox-active iron is bound to neuromelanin and thereby retained within this compartment, but in Parkinson's disease it is thought to be increasingly released into the cytosol, promoting oxidative stress. This unit includes a methodological workflow for the isolation of ... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=2042815 Mon, 01 Dec 2008 05:00:00 +0100 2042815 http://www.medworm.com/index.php?rid=2042814&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D19085989%26dopt%3DAbstract Authors: Shroff H, White H, Betzig E Key to understanding a protein's biological function is the accurate determination of its spatial distribution inside a cell. Although fluorescent protein markers allow the targeting of specific proteins with molecular precision, much of this information is lost when the resultant fusion proteins are imaged with conventional, diffraction-limited optics. In response, several imaging modalities that are capable of resolution below the diffraction limit ( approximately 200 nm) have emerged. Here, both single- and dual-color superresolution imaging of biological structures using photoactivated localization microscopy (PALM) are described. The examples discussed focus on adhesion complexes: dense, protein-filled assemblies that form at the interface betw... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=2042814 Mon, 01 Dec 2008 05:00:00 +0100 2042814 http://www.medworm.com/index.php?rid=1834002&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18819087%26dopt%3DAbstract Authors: Serban MA, Scott A, Prestwich GD The practice of in vitro three-dimensional (3-D) cell culture has lagged behind the realization that classical two-dimensional (2-D) culture on plastic surfaces fails to mirror normal cell biology. Biologically, a complex network of proteins and proteoglycans that constitute the extracellular matrix (ECM) surrounds every cell. To recapitulate the normal cellular behavior, scaffolds (ECM analogs) that reconstitute the essential biological cues are required. This unit describes the 3-D cell culture and tumor engineering applications of Extracel, a novel semisynthetic ECM (sECM), based on cross-linked derivatives of hyaluronan and gelatin. A simplified cell encapsulation and pseudo-3-D culturing (on top of hydrogels) protocol is provided. In addit... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1834002 Mon, 01 Sep 2008 04:00:00 +0100 1834002 http://www.medworm.com/index.php?rid=1834001&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18819088%26dopt%3DAbstract Authors: Birkedal-Hansen H, Yamada S, Windsor J, Pollard AH, Lyons G, Stetler-Stevenson W, Birkedal-Hansen B Matrix metalloproteinases are a class of enzymes that play an important role in the remodeling of the extracellular matrix in development and cancer metastasis. This unit describes a set of methods--cell-mediated dissolution of type-1 collagen fibrils, direct and reverse zymography, enzyme capture based on alpha2-macroglobulin and TIMP-1 and -2, and demonstration of cryptic thiol groups in metalloproteinase precursors--that are used to characterize the functions of matrix metalloproteinases and their inhibitors. PMID: 18819088 [PubMed - in process] (Source: Current Protocols in Cell Biology) Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1834001 Mon, 01 Sep 2008 04:00:00 +0100 1834001 http://www.medworm.com/index.php?rid=1834000&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18819089%26dopt%3DAbstract Authors: Barriere H, Lukacs GL The post-endocytic sorting of internalized membrane proteins plays a critical role in numerous physiological processes, including receptor desensitization, degradation of non-native plasma membrane proteins, and cell surface retrieval of receptors from early endosomes upon ligand dissociation. Here, we describe a fluorescence ratiometric image analysis (FRIA) method used to determine the post-endocytic fate and transport kinetics of transmembrane proteins based on the pH measurement of internalized cargo-containing compartments in living cells. The method relies on the notion that the pH of a cargo-containing transport vesicle (vesicular pH, pH(v)) could be taken as an indicator of its identity, considering that endocytic organelles (e.g., sorting endosom... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1834000 Mon, 01 Sep 2008 04:00:00 +0100 1834000 http://www.medworm.com/index.php?rid=1833999&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18819090%26dopt%3DAbstract Authors: Manley S, Gordon VD This unit describes protocols for making giant unilamellar vesicles (GUVs) based on rehydration of dried lipid films. These model membranes are useful for determining the impact of membrane and membrane-binding components on lipid bilayer stiffness and phase behavior. Due to their large size, they are especially amenable to studies using fluorescence and light microscopy, and may also be manipulated for mechanical measurements with optical traps or micropipets. In addition to their use in encapsulation, GUVs have proven to be useful model systems for studying many cellular processes, including tubulation, budding, and fusion, as well as peptide insertion. The introduction of enzymes or proteins can result in reorganization, leading to such diverse behavior ... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1833999 Mon, 01 Sep 2008 04:00:00 +0100 1833999 http://www.medworm.com/index.php?rid=1833998&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18819091%26dopt%3DAbstract Authors: Seigneurin-Berny D, Salvi D, Joyard J, Rolland N Chloroplasts are plant-specific organelles. They are the site of photosynthesis but also of many other essential metabolic pathways, such as syntheses of amino acids, vitamins, lipids, and pigments. This unit describes the isolation and purification of chloroplasts from Arabidopsis and spinach leaves. Differential centrifugation is first used to obtain a suspension enriched in chloroplasts (crude chloroplasts extract). In a second step, Percoll density gradient centrifugation is used to recover pure and intact chloroplasts. The Basic Protocol describes the purification of chloroplasts from Arabidopsis leaves. This small flowering plant is now widely used as a model organism in plant biology as it offers important advantages for ... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1833998 Mon, 01 Sep 2008 04:00:00 +0100 1833998 http://www.medworm.com/index.php?rid=1817790&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18551418%26dopt%3DAbstract Authors: Pestov DG, Lapik YR, Lau LF The synthesis of ribosomes is a major metabolic activity critical for cell growth and homeostasis. Understanding the mechanisms of ribosome biogenesis has important implications for studying both protein synthesis and cell cycle control. This unit describes several techniques for the analysis of rRNA maturation and ribosome assembly adapted for mammalian cells. Metabolic labeling of rRNA and hybridization analysis of precursors can be used to assess changes in rRNA processing that occur under experimental conditions of interest. Separation of preribosomal particles by sucrose gradient centrifugation is suitable for the analysis of proteins associated with preribosomes during their assembly and maturation in the cell nucleus. PMID: 18551418 [PubM... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817790 Sun, 01 Jun 2008 04:00:00 +0100 1817790 http://www.medworm.com/index.php?rid=1817789&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18551419%26dopt%3DAbstract Authors: Schermelleh L, Spada F, Leonhardt H In this unit, a live-cell assay to measure DNA (cytosine-5) methyltransferase (MTase) activity at the single-cell level is described. This method takes advantage of the irreversible binding of enzymatically active MTases to genomic DNA substituted with the mechanism-based inhibitor 5-aza-2'-deoxycytidine (5-aza-dC). The procedure comprises incorporation of this nucleoside analog into DNA during replication and quantification of the time-dependent MTase immobilization by fluorescence recovery after photobleaching (FRAP). This trapping assay monitors kinetic properties and activity-dependent immobilization of MTases in their native environment and enables direct comparison of mutations and inhibitors that affect MTase regulation and catalytic ... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817789 Sun, 01 Jun 2008 04:00:00 +0100 1817789 http://www.medworm.com/index.php?rid=1817788&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18551420%26dopt%3DAbstract Authors: Avila J, Soares H, Fanarraga ML, Zabala JC This unit describes various protocols for the isolation and purification of the main constituents of microtubules, chiefly alpha- and beta-tubulin, and the most significant microtubule associated proteins (MAPs), specifically MAP1A, MAP1B, MAP2, and tau. We include a classical isolation method for soluble tubulin heterodimer as the first basic purification protocol. In addition, we show how to analyze the tubulin and MAPs obtained after a phosphocellulose chromatography purification procedure. This unit also details a powerful and simple method to determine the native state of the purified tubulin based on one-dimensional electrophoresis under nondenaturing conditions (UNIT 6.5). The last protocol describes the application of a new te... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817788 Sun, 01 Jun 2008 04:00:00 +0100 1817788 http://www.medworm.com/index.php?rid=1817787&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18551421%26dopt%3DAbstract Authors: Mavrakis M, Rikhy R, Lilly M, Lippincott-Schwartz J This unit describes fluorescence-based techniques for noninvasive imaging of development in living Drosophila embryos, discussing considerations for fluorescent imaging within living embryos and providing protocols for generation of flies expressing fluorescently tagged proteins and for preparation of embryos for fluorescent imaging. The unit details time-lapse confocal imaging of live embryos and discusses optimizing image acquisition and performing three-dimensional imaging. Finally, the unit provides a variety of specific methods for optical highlighting of specific subsets of fluorescently tagged proteins and organelles in the embryo, including fluorescence recovery after photobleaching (FRAP), fluorescence loss in photob... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817787 Sun, 01 Jun 2008 04:00:00 +0100 1817787 http://www.medworm.com/index.php?rid=1817786&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18551422%26dopt%3DAbstract Authors: Zinchuk V, Zinchuk O Colocalization is an important finding in many cell biological studies. This unit describes a protocol for quantitative evaluation of images with colocalization based on the calculation of a number of specialized coefficients. First, images of double-stained sections are subjected to background correction. Then, various coefficients are calculated. Meanings of the coefficients and a guide to interpretation of their results indicating either presence or absence of colocalization are given. Success in colocalization studies depends on the quality of analyzed images, proper preparation of them for coefficients calculations, and correct interpretation of obtained results. This protocol helps to ensure reliability of colocalization coefficients calculations. ... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817786 Sun, 01 Jun 2008 04:00:00 +0100 1817786 http://www.medworm.com/index.php?rid=1817785&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18551423%26dopt%3DAbstract Authors: Jedeszko C, Sameni M, Olive MB, Moin K, Sloane BF Proteolytic degradation of extracellular matrix (ECM) components by cells is an important metabolic activity as cells grow, remodel, and migrate through the ECM. The ability to analyze ECM degradation can be valuable in the study of developmental processes as well as pathologies, such as cancer. In this unit we describe an in vitro live cell-based method to image and quantitatively measure the degradation of ECM components by live cells. Cells are grown in the presence of fluorescent dye-quenched protein substrates (DQ-gelatin, DQ-collagen I, and DQ-collagen IV) that are mixed with protein matrices. Upon proteolytic cleavage, fluorescence is released that directly reflects the level of proteolysis by the cells. Using confocal m... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817785 Sun, 01 Jun 2008 04:00:00 +0100 1817785 http://www.medworm.com/index.php?rid=1817794&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18360815%26dopt%3DAbstract Authors: Pellegrin S, Mellor H The Rho GTPase family of signaling proteins controls a wide range of highly dynamic cellular processes. Activation of Rho GTPases can be investigated and quantified in cell extracts using so-called pull-down assays. Proteins that bind specifically to the activated form of the Rho GTPase are used to capture it onto a bead support. Western blotting of the captured samples with specific antibodies then allows for quantification of the level of Rho GTPase activation in the sample. This unit describes the techniques for preparing the reagents required for assays of RhoA, Rac, and Cdc42 and gives practical tips for the successful application of the assay in a range of situations. PMID: 18360815 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Ce... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817794 Sat, 01 Mar 2008 05:00:00 +0100 1817794 http://www.medworm.com/index.php?rid=1817793&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18360816%26dopt%3DAbstract Authors: Patterson GH A major advance in the microscopic study of cells and tissues is the introduction of photoactivatable fluorescent proteins, which can specifically mark proteins of interest within a living cell. Fluorescent proteins are now available that allow a pool of molecules to be "turned on" by photoactivation. This unit discusses technical aspects for the general use of photoactivatable fluorescent proteins and introduces some specific applications in the concluding remarks. PMID: 18360816 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Cell Biology) Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817793 Sat, 01 Mar 2008 05:00:00 +0100 1817793 http://www.medworm.com/index.php?rid=1817792&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18360817%26dopt%3DAbstract Authors: Denyer K, Pike M Two different methods for the preparation of starch-rich plastids are described together with protocols for the determination of plastid yield, purity, and intactness. The preparation of amyloplasts from maize endosperm and oilseed rape embryos are given as examples, but the protocols could be adapted for the isolation of starch-rich plastids from other plant organs. A method for the determination of the quantitative distribution of an enzyme between the plastids and cytosol is given. Typical results and references for marker enzymes for a range of subcellular compartments are listed. PMID: 18360817 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Cell Biology) Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817792 Sat, 01 Mar 2008 05:00:00 +0100 1817792 http://www.medworm.com/index.php?rid=1817791&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18360818%26dopt%3DAbstract Authors: Schamel WW Multiprotein complexes play crucial roles in nearly all cell biological processes. Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) is a powerful method to study these complexes. It is a native protein separation method that relies on the dye Coomassie blue to confer negative charge for separation. It has a higher resolution than gel filtration or sucrose density ultracentrifugation and can be used for protein complexes from 10 kDa to 10 MDa. If a second-dimension SDS-PAGE is applied (two-dimensional BN/SDS-PAGE), the size, subunit composition, and relative abundance of the different multiprotein complexes can be studied. In recent years, there has been a large increase in the number of publications where BN-PAGE was used to study protein-protein interaction... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817791 Sat, 01 Mar 2008 05:00:00 +0100 1817791 http://www.medworm.com/index.php?rid=1817813&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228500%26dopt%3DAbstract Authors: Valencia-Burton M, Broude NE This unit describes a method allowing RNA visualization in live cells. The method is based on fluorescent protein complementation regulated by RNA-aptamer/RNA-binding protein interactions. Based on these two principles, a fluorescent ribonucleoprotein complex is assembled inside the cell only in response to the presence of the aptamer sequence on the target RNA. PMID: 18228500 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Cell Biology) Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817813 Sat, 01 Dec 2007 05:00:00 +0100 1817813 http://www.medworm.com/index.php?rid=1817801&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228512%26dopt%3DAbstract Authors: Buck CB, Thompson CD Papillomaviruses are a diverse group of pathogens that infect the skin and mucosal tissues of humans and various animal species. The viral genome is a circular, double-stranded DNA molecule approximately 8-kb in length. The non-enveloped papillomavirus capsid is composed of a virally encoded major coat protein, L1, and a minor coat protein, L2. L1 and L2 co-assemble when expressed in mammalian cells, and can promiscuously encapsidate essentially any <8-kb plasmid present in the cell nucleus. In the last several years, there has been rapid development of techniques for intracellular production of papillomavirus-based gene transfer vectors (also known as pseudoviruses). This unit outlines the production and propagative amplification of papillomaviral vect... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817801 Sat, 01 Dec 2007 05:00:00 +0100 1817801 http://www.medworm.com/index.php?rid=1817798&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228515%26dopt%3DAbstract Authors: Bozidis P, Williamson CD, Colberg-Poley AM Increasingly mechanistic virology studies require dependable and sensitive methods for isolating purified organelles containing functional cellular sub-domains. The mitochondrial network is, in part, closely apposed to the endoplasmic reticulum (ER). The mitochondria-associated membrane (MAM) fraction provides direct physical contact between the ER and mitochondria. Characterization of the dual localization and trafficking of human cytomegalovirus (HCMV) UL37 proteins required establishing protocols in which the ER and mitochondria could be reliably separated. Because of its documented role in lipid and ceramide transfer from the ER to mitochondria, a method to purify MAM from infected cells was also developed. Two robust procedures w... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817798 Sat, 01 Dec 2007 05:00:00 +0100 1817798 http://www.medworm.com/index.php?rid=1817796&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228517%26dopt%3DAbstract Authors: Passey S, Pellegrin S, Mellor H The surface of metazoan cells is a landscape not clearly visualized by light microscopy. Many cells elaborate protrusive structures such as microvilli, filopodia, lamellipodia, and surface ruffles that play important roles in the interaction between the cell and its environment. The high resolution of scanning electron microscopy makes it an ideal technique for studies of the cell surface; however, preservation of fine surface structure can be problematic. Here we highlight the critical factors in sample preparation to ensure optimal high-resolution imaging of the surface of mammalian cells and tissues. PMID: 18228517 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Cell Biology) Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817796 Sat, 01 Dec 2007 05:00:00 +0100 1817796 http://www.medworm.com/index.php?rid=1817795&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228518%26dopt%3DAbstract Authors: Gallagher SR Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to investigate subunit compositions, and to verify homogeneity of protein samples. It can also serve to purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in a polyacrylamide gel matrix; pore size decreases with increasing acrylamide concentration. The combination of pore size and protein charge, size, and shape determines the migration rate of the protein. In this unit, the standard Laemmli method is described for discontinuous gel electrophoresis under denaturing conditions, that is, in the presence of sodium dodec... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817795 Sat, 01 Dec 2007 05:00:00 +0100 1817795 http://www.medworm.com/index.php?rid=1817819&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228494%26dopt%3DAbstract Authors: Phelan MC Cultured mammalian cells are used extensively in cell biology studies. It requires a number of special skills in order to be able to preserve the structure, function, behavior, and biology of the cells in culture. This unit describes the basic skills required to maintain and preserve cell cultures: maintaining aseptic technique, preparing media with the appropriate characteristics, passaging, freezing and storage, recovering frozen stocks, and counting viable cells. PMID: 18228494 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Cell Biology) Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817819 Sat, 01 Sep 2007 04:00:00 +0100 1817819 http://www.medworm.com/index.php?rid=1817815&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228498%26dopt%3DAbstract Authors: Taupenot L The catecholamine-secreting PC12 cell line derived from the rat adrenal medulla has long been considered a model system for neurosecretion and neuronal differentiation. PC12 cells contain a large number of secretory granules (otherwise known as large dense-core vesicles) for storage of small molecules, processing enzymes, neuropeptides, and peptide hormones. Secretory granule exocytosis in PC12 cells is tightly regulated by calcium and occurs in response to a secretagogue. This unit provides protocols for maintenance and transfection of PC12 cells. Several secretion assays are described to measure the release of secretory granule cargo molecules by detection of radioactive catecholamine, or by immunochemical or chemiluminescence detection of transfected regulated se... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817815 Sat, 01 Sep 2007 04:00:00 +0100 1817815 http://www.medworm.com/index.php?rid=1817811&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228502%26dopt%3DAbstract Authors: Olenych SG, Claxton NS, Ottenberg GK, Davidson MW Advances in fluorescent protein development over the past 10 years have led to fine-tuning of the Aequorea victoria jellyfish color palette in the emission color range from blue to yellow, while a significant amount of progress has been achieved with reef coral species in the generation of monomeric fluorescent proteins emitting in the orange to far-red spectral regions. It is not inconceivable that near-infrared fluorescent proteins loom on the horizon. Expansion of the fluorescent protein family to include optical highlighters and FRET biosensors further arms this ubiquitous class of fluorophores with biological probes capable of photoactivation, photoconversion, and detection of molecular interactions beyond the resolution l... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817811 Sat, 01 Sep 2007 04:00:00 +0100 1817811 http://www.medworm.com/index.php?rid=1817806&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228507%26dopt%3DAbstract Authors: Vodyanik MA, Slukvin II Human embryonic stem cells (hESCs) represent a unique population of cells capable of self-renewal and differentiation into all types of somatic cells, including hematopoietic and endothelial cells. Since the pattern of hematopoietic and endothelial development observed in the embryo can be reproduced using ESCs differentiated in culture, hESCs can be used as a model for studies of specification and diversification of hematoendothelial progenitors. In addition, hESCs can be seen as a scalable source of hematopoietic and endothelial cells for in vitro studies. This unit describes a method for efficient differentiation of hESCs into hematopoietic progenitors and endothelial cells through coculture with mouse OP9 bone marrow stromal cells, as well as an app... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817806 Sat, 01 Sep 2007 04:00:00 +0100 1817806 http://www.medworm.com/index.php?rid=1817805&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228508%26dopt%3DAbstract Authors: Cohen MA, Itsykson P, Reubinoff BE Human embryonic stem cells (hESCs) may be converted into highly enriched cultures of neural precursors under defined culture conditions. The neural precursors can proliferate in culture for prolonged periods of time, and can differentiate in vitro into mature neurons, astrocytes, and oligodendrocytes. The neurons are functional and have normal electrophysiological properties. After transplantation to the developing rodent brain, the neural precursors migrate extensively into the host brain parenchyma, respond to host brain signals, and differentiate in a region-specific manner to progeny of the three neural lineages. The establishment of neuroectodermal precursors from hESCs allows the study of human neurogenesis in vitro and is an aid in dru... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817805 Sat, 01 Sep 2007 04:00:00 +0100 1817805 http://www.medworm.com/index.php?rid=1817802&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228511%26dopt%3DAbstract Authors: S Lidke D, Nagy P, J Arndt-Jovin D This unit describes the use of quantum dots (QDs) for live-cell imaging and the use of QDs in flow cytometry for quantitative analysis of ligand binding constants and receptor density. Conventional fluorophores and visible fluorescent protein (VFP) constructs have allowed visualization of many cellular processes. However, organic and biomolecular fluorophores have limitations in their applications, due to their small Stokes' shift and tendency to photobleach during prolonged imaging. QDs have many advantages over conventional fluorophores, including high brightness and photostability, which make them an exceptional tool for live-cell imaging. There are a large variety of commercially available QDs with different surface reactivities and chara... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817802 Sat, 01 Sep 2007 04:00:00 +0100 1817802 http://www.medworm.com/index.php?rid=1817816&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228497%26dopt%3DAbstract Authors: Hamilton N, Kerr MC, Burrage K, Teasdale RD With the advent of live cell imaging microscopy, new types of mathematical analyses and measurements are possible. Many of the real-time movies of cellular processes are visually very compelling, but elementary analysis of changes over time of quantities such as surface area and volume often show that there is more to the data than meets the eye. This unit outlines a geometric modeling methodology and applies it to tubulation of vesicles during endocytosis. Using these principles, it has been possible to build better qualitative and quantitative understandings of the systems observed, as well as to make predictions about quantities such as ligand or solute concentration, vesicle pH, and membrane trafficked. The purpose is to outline ... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817816 Fri, 01 Jun 2007 04:00:00 +0100 1817816 http://www.medworm.com/index.php?rid=1817812&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228501%26dopt%3DAbstract Authors: Kedrin D, Wyckoff J, Sahai E, Condeelis J, Segall JE This unit describes the methods that we have been developing for analyzing tumor cell motility in mouse and rat models of breast cancer metastasis. Rodents are commonly used to provide a mammalian system for studying human tumor cells as xenografts in immunocompromised mice, as well as for following the development of tumors from a specific tissue type in transgenic lines. The Basic Protocol describes the standard methods used for generation of mammary tumors and imaging them. Additional protocols for labeling macrophages, blood vessel imaging, and image analysis are also included. PMID: 18228501 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Cell Biology) Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817812 Fri, 01 Jun 2007 04:00:00 +0100 1817812 http://www.medworm.com/index.php?rid=1817810&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228503%26dopt%3DAbstract Authors: Yokochi T, Gilbert DM In this unit, several basic protocols to identify sites of DNA replication utilizing incorporation of halogenated thymidine analogs into DNA, followed by immunofluorescent imaging are described. Antibodies specific for halogenated thymidine analogs such as bromodeoxyuridine (BrdU), chlorodeoxyuridine (CldU), and iododeoxyuridine (IdU) can provide a rapid, nonhazardous, and sensitive method for detecting DNA replication in single cells, in a manner analogous to the traditional use of tritiated thymidine. In combination with different techniques to prepare the DNA template, a variety of DNA replication-related events can be examined by conventional fluorescence-microscopic approaches. Because origin firing and the progression of replication forks are regula... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817810 Fri, 01 Jun 2007 04:00:00 +0100 1817810 http://www.medworm.com/index.php?rid=1817804&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228509%26dopt%3DAbstract Authors: Singh RD, Marks DL, Pagano RE Sphingolipids (SLs), including glycosphingolipids, are found on the plasma membrane where they play important roles in a wide variety of cell functions, including cell-cell communication, cell growth and differentiation, host-pathogen interactions, and cell-signaling events. This unit illustrates the use of fluorescent SL analogs to identify the mechanisms underlying SL endocytosis and subsequent intracellular trafficking. Techniques used to study SL domain formation at the plasma membrane, endocytic mechanisms and intracellular transport steps are highlighted. The use of biochemical treatments and dominant-negative protein expression to block specific steps in lipid trafficking are also discussed. PMID: 18228509 [PubMed - indexed for MEDLINE]... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817804 Fri, 01 Jun 2007 04:00:00 +0100 1817804 http://www.medworm.com/index.php?rid=1817803&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228510%26dopt%3DAbstract Authors: Listenberger LL, Brown DA Most eukaryotic cells can store excess lipid in cytosolic lipid droplets. This unit discusses techniques for the visualization of lipid droplets and associated proteins in cultured mammalian cells. Protocols for the detection of lipid droplets with nile red and BODIPY 493/503 are included. The differences in the spectral properties of these two lipophilic dyes and advantages of each are discussed. The best method for combining visualization of intracellular lipid droplets with indirect immunofluorescent detection of lipid droplet-associated proteins is described. Techniques for sample fixation and permeabilization must be chosen carefully to avoid alterations to lipid droplet morphology. Immunofluorescent detection of adipophilin, a broadly expressed,... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817803 Fri, 01 Jun 2007 04:00:00 +0100 1817803 http://www.medworm.com/index.php?rid=1817817&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228496%26dopt%3DAbstract Authors: Stuart RA, Koehler CM This unit describes methods for importing in vitro-translated or recombinant proteins into isolated yeast mitochondria and for exporting mitochondrial proteins translated in the yeast mitochondrial matrix into the inner mitochondrial membrane. The methods use mitochondria isolated from yeast cells and mitochondrial protein precursors derived from an in vitro transcription/translation reaction or purified from an E. coli recombinant protein expression system. The described translocation assays can be used to determine whether a protein is targeted to mitochondria and its location within the mitochondrion. It can also be used to study the import mechanism and to investigate mitochondrial matrix translation of proteins and their export. PMID: 18228496 [P... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817817 Thu, 01 Mar 2007 05:00:00 +0100 1817817 http://www.medworm.com/index.php?rid=1817809&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228504%26dopt%3DAbstract Authors: Kappas NC, Bautch VL Embryonic stem (ES) cells, which are derived from developing mouse blastocysts, have the capacity to give rise to all cell types in the adult body. The ability of ES cells to do so has opened the door for novel experimental approaches in the field of developmental biology. Under appropriate culture conditions, ES cells will differentiate and form embryoid bodies (EBs). Upon attachment to a permissive surface, EBs continue a programmed differentiation, and many of the cells differentiated from the EBs reflect those found in the developing embryo and yolk sac, such as hematopoietic cells, endoderm, and endothelial cells. Endothelial cells that arise during ES cell differentiation have the potential to form primitive blood vessels, comparable to the vessels t... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817809 Thu, 01 Mar 2007 05:00:00 +0100 1817809 http://www.medworm.com/index.php?rid=1817808&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228505%26dopt%3DAbstract Authors: Wdziekonski B, Villageois P, Dani C The authors describe protocols for culture conditions in which mouse ES cells can be maintained in an undifferentiated state or committed to undergo adipocyte differentiation at a high rate and in a highly reproducible fashion. There is also a protocol for maintaining and differentiating human adult stem cells, isolated form adipose tissue and from bone marrow, into adipocytes. These culture systems provide a powerful means for studying the first step of adipose cell development and a means to investigate effects of drugs on the biology of adipocytes. There are also protocols for detection of adipocytes and analysis of their gene expression. PMID: 18228505 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Cell Biology) Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817808 Thu, 01 Mar 2007 05:00:00 +0100 1817808 http://www.medworm.com/index.php?rid=1817807&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228506%26dopt%3DAbstract Authors: Kramer J, Schlenke P, Rohwedel J This unit describes the protocols used for cultivation of murine embryonic stem (ES) cells and their differentiation into chondrogenic cell types in vitro. ES cells cultivated as cellular aggregates, so-called embryoid bodies (EBs), differentiate spontaneously into chondrogenic cell types recapitulating cellular events of chondro- and osteogenesis. The undifferentiated ES cells differentiate into mesenchymal prechondrogenic cells in the EB outgrowths. These progenitor cells aggregate and form mesenchymal condensations. During further cultivation, these cells form cartilage nodules, show a phenotype typical for chondroblasts, and start to express marker molecules of cartilage tissue. Later, the chondrocytes become hypertrophic, and finally, mark... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817807 Thu, 01 Mar 2007 05:00:00 +0100 1817807 http://www.medworm.com/index.php?rid=1817799&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228514%26dopt%3DAbstract Authors: Morré DJ, Hammond T Methods are described to isolate intact brush borders and brush border membranes from renal cell homogenates. A rapid method yields sealed vesicles that reconstitute renal brush border transport. In one variation of this protocol, 10 to 20 mM CaCl2 or MgCl2 is added to aggregate non-brush border structures for subsequent removal by centrifugation. For analytical studies, guidance is provided for subsequent purification steps including preparative free-flow and aqueous two-phase partition. Marker enzymes and morphological parameters are included for assessment of yield and fraction purity. PMID: 18228514 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Cell Biology) Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817799 Thu, 01 Mar 2007 05:00:00 +0100 1817799 http://www.medworm.com/index.php?rid=1817797&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228516%26dopt%3DAbstract Authors: Friedl P, Wolf K, von Andrian UH, Harms G Multiphoton microscopy has become a standard method for noninvasive imaging of thick specimens with subcellular resolution. Higher harmonic generation microscopy (HHGM), based on nonlinear multiphoton excitation, is a contrast mechanism for the structural and molecular imaging of native samples in cell culture and in fixed and live tissues, for both, three-dimensional and four-dimensional reconstructions. HHGM comprises second and third harmonic generation (SHG, THG) of ordered molecules, can be obtained without exogenous labels, and provides detailed real-time optical reconstruction of fibrillar collagen, myosin, microtubules, and membrane potential, as well as cell depolarization. This unit presents the principles of SHG and THG and ... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817797 Thu, 01 Mar 2007 05:00:00 +0100 1817797 http://www.medworm.com/index.php?rid=1817818&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228495%26dopt%3DAbstract Authors: Beacham DA, Amatangelo MD, Cukierman E Fibroblasts secrete and organize extracellular matrix (ECM), which provides structural support for their adhesion, migration, and tissue organization, besides regulating cellular functions such as growth and survival. Cell-to-matrix interactions are vital for vertebrate development. Disorders in these processes have been associated with fibrosis, developmental malformations, cancer, and other diseases. This unit describes a method for preparing a three-dimensional matrix derived from NIH-3T3 cells; the matrix is three-dimensions, cell and debris free, and attached to a two-dimensional culture surface. Cell adhesion and spreading are normal on these matrices. This matrix can also be compressed into a two-dimensional matrix and solubilized ... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817818 Mon, 01 Jan 2007 05:00:00 +0100 1817818 http://www.medworm.com/index.php?rid=1817814&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228499%26dopt%3DAbstract Authors: Melcher K, Chen HT Chemical crosslinking provides information about protein-protein interactions in the context of intact protein complexes; therefore, it is particularly suited to the analysis of multiprotein complexes. Rather than a single distinct technique, chemical crosslinking represents a smorgasbord of techniques that differ significantly both in chemistry and in scope. This unit will attempt to guide the reader through the complexities of crosslinking to find the most suitable approach for a given biological question. Sample protocols for two crosslinking methods considered to be particularly useful for the analysis of large multiprotein complexes are provided: His6-mediated crosslinking and photoinducible label transfer crosslinking. PMID: 18228499 [PubMed - inde... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817814 Mon, 01 Jan 2007 05:00:00 +0100 1817814 http://www.medworm.com/index.php?rid=1817800&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228513%26dopt%3DAbstract Authors: Larocca JN, Norton WT The methods used to prepare myelin involve homogenization of the tissue in isotonic sucrose solution, followed by the isolation of myelin membranes by a series of steps that include density gradient centrifugation and differential centrifugation. Homogenization of nervous tissue in isotonic sucrose causes the myelin sheath to peel from the axon and form relatively large myelin vesicles. The large size of the myelin vesicles, together with the fact that myelin membrane has a lower density than other biological membranes, make differential centrifugation and density gradient centrifugation the main tools for the isolation of this membrane. Three protocols are outlined in this unit: isolation of a highly-purified myelin fraction from the central nervous syst... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817800 Mon, 01 Jan 2007 05:00:00 +0100 1817800 http://www.medworm.com/index.php?rid=1817836&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228477%26dopt%3DAbstract Authors: Amessou M, Popoff V, Yelamos B, Saint-Pol A, Johannes L The recently described retrograde transport route is a highly selective pathway that allows some internalized molecules to reach the trans-Golgi network from early/recycling endosomes, bypassing the recycling route to the plasma membrane and the late endocytic pathway. The non-toxic receptor-binding B-subunit of bacterial Shiga toxin has played an important role in the discovery and molecular dissection of membrane trafficking at the early/recycling endosomes-TGN interface. This unit describes several recent methods for quantitative biochemical and morphological analysis of retrograde transport. The sulfation assay permits the detection and quantification of cargo protein transport from endosomes to the TGN, describing ho... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817836 Sun, 01 Oct 2006 04:00:00 +0100 1817836 http://www.medworm.com/index.php?rid=1817835&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228478%26dopt%3DAbstract Authors: Blank U, Rivera J Mast cells are important effectors in innate and adaptive immune responses. They contain numerous secretory granules filled with inflammatory mediators in their cytoplasm. Exocytosis of granular content does not take place until the cell receives an appropriate stimulus such as the aggregation of IgE antibody bound to high-affinity IgE receptors by specific antigen. This process is therefore referred to as regulated exocytosis. A characteristic of mast cell exocytosis is that it does not involve release of a few individual granules, but rather, a large fraction of the granular content is released due to compound exocytosis, implicating the occurrence of granule-to-granule and granule-to-plasma membrane fusion. This unit describes assays that measure the relea... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817835 Sun, 01 Oct 2006 04:00:00 +0100 1817835 http://www.medworm.com/index.php?rid=1817833&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228480%26dopt%3DAbstract Authors: Snapp EL, Hegde RS Fluorescence resonance energy transfer (FRET) refers to the nonradiative transfer of energy from one fluorescent molecule (the donor) to another fluorescent molecule (the acceptor). Measurement of FRET between two fluorophore-labeled proteins can be used to infer the subnanometer spatial and temporal characteristics of protein interactions in their native cellular environment. Multiple experimental methods exist for measuring FRET. The method that can be most widely and simply implemented, quantified, and interpreted is the acceptor-photobleaching FRET technique. In this method, the presence of FRET between a donor and acceptor is revealed upon destruction (by photobleaching) of the acceptor. Acceptor photobleaching can be exploited to detect changes in the ... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817833 Sun, 01 Oct 2006 04:00:00 +0100 1817833 http://www.medworm.com/index.php?rid=1817832&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228481%26dopt%3DAbstract Authors: Viviani B The close relationship between neurons, glia, astroglia, and astrocytes plays a relevant role in modulating a neurotoxic effect and propagating the consequent degenerative event in the brain. After exposure to a neurotoxicant, glial cells are often activated to release substances responsible for the propagation of the damage. the production of such mediators is the result of both neural death and communication between microglia and astrocytes. Because they are simple and highly controlled, cocultures of different cells of the nervous system are a valuable tool in the investigations of these complex relationships. This unit describes protocols to set up neuron-glia and microglia-astrocyte sandwich cocultures, in which the selected cell populations grow on two differen... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817832 Sun, 01 Oct 2006 04:00:00 +0100 1817832 http://www.medworm.com/index.php?rid=1817834&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228479%26dopt%3DAbstract Authors: Lorick KL, Yang Y, Jensen JP, Iwai K, Weissman AM A concept that has arisen over the last decade is that proteins can, in general, be covalently modified by polypeptides, resulting in alterations in their fate and function. The first-identified and most well studied of these modifying polypeptides is ubiquitin. Although targeting for proteasomal degradation is the best studied outcome of ubiquitylation, we now understand that modification of proteins with ubiquitin has numerous other cellular roles that alter protein function and that are unrelated to proteasomal degradation. Ubiquitylation is a complex process that is regulated at the level of both addition and removal of ubiquitin from target proteins. This unit includes a number of different basic protocols that will facili... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817834 Sat, 01 Jul 2006 04:00:00 +0100 1817834 http://www.medworm.com/index.php?rid=1817822&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228491%26dopt%3DAbstract Authors: Leung CL, Liem RK Intermediate filaments (IFs) are found in most eukaryotic cells and are made up of various IF proteins. IFs are highly insoluble in conventional extraction buffers and are therefore commonly purified under denaturing condition. Purified IF proteins can be reassembled into filaments by dialysis. At least 65 IF proteins are found in humans, and the procedures for the purification of each subunit vary somewhat, although many basic steps are similar. To illustrate the isolation of IFs, a detailed protocol is described for purifying neurofilament proteins (NFL, NFM, and NFH subunits) from bovine spinal cord. These three proteins form the predominant IF network in mature neurons. An alternative method for the purification of NFL from a prokaryotic expression system... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817822 Sat, 01 Jul 2006 04:00:00 +0100 1817822 http://www.medworm.com/index.php?rid=1817821&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228492%26dopt%3DAbstract Authors: Zorzano A, Camps M The transverse tubules (T-tubules) of mammalian cardiac and skeletal muscles are invaginations of the sarcolemma. They play a crucial role in excitation-contraction coupling as well as in intracellular signaling and in regulation of glucose transport. The biochemical purification of T-tubule membranes is a difficult task, and membrane fractions enriched in transverse tubules are usually contaminated with other cell-surface and intracellular membranes. This unit includes methods that permit the isolation and purification of T-tubules from skeletal muscle. PMID: 18228492 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Cell Biology) Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817821 Sat, 01 Jul 2006 04:00:00 +0100 1817821 http://www.medworm.com/index.php?rid=1817837&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228476%26dopt%3DAbstract Authors: Bernard-Trifilo JA, Lim ST, Hou S, Schlaepfer DD, Ilic D Integrins are a family of heterodimeric alpha/beta transmembrane cell adhesion receptors that play important roles in the regulation of cell migration, proliferation, and survival. Integrins do not possess intrinsic catalytic activity, and signaling events are mediated by their lateral association with other cell surface receptors or clustering of their cytoplasmic domains with signaling proteins. Rapid activation of protein-tyrosine kinases is one of the first signaling events associated with integrin binding to the extracellular matrix protein fibronectin. The intracellular focal adhesion kinase (FAK) is recruited to sites of integrin clustering, and this unit describes the methods with which to analyze FAK phosphoryla... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817837 Sat, 01 Apr 2006 05:00:00 +0100 1817837 http://www.medworm.com/index.php?rid=1817825&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228488%26dopt%3DAbstract Authors: Kandror KV, Pilch PF The immunoisolation of GLUT4-containing vesicles from adipocytes is described in this unit. The methods involve homogenization of cells followed by differential centrifugation to provide the intracellular membranes that contain GLUT4. Subsequently, an immobilized monoclonal antibody is used for the isolation of vesicles of very high purity. The various protocols are applicable to cultured and primary adipocytes as well as skeletal muscle, the major insulin target cells expressing GLUT4. PMID: 18228488 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Cell Biology) Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817825 Sat, 01 Apr 2006 05:00:00 +0100 1817825 http://www.medworm.com/index.php?rid=1817824&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228489%26dopt%3DAbstract Authors: Cheeseman CI, O'Neill D This unit provides protocols for isolating intestinal brush-border membranes from rat, pig, and cow. These membranes can be used for immunoblotting or other analytical techniques. They will also spontaneously form closed vesicles which allow for flux assays to be performed using rapid filtration techniques. Overall the isolation procedures take approximately 3.5 hr. The resulting isolated membranes can be stored under liquid nitrogen or at -70 degrees C for a week or more depending upon the species. PMID: 18228489 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Cell Biology) Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817824 Sat, 01 Apr 2006 05:00:00 +0100 1817824 http://www.medworm.com/index.php?rid=1817823&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228490%26dopt%3DAbstract Authors: Théry C, Amigorena S, Raposo G, Clayton A Exosomes are small membrane vesicles found in cell culture supernatants and in different biological fluids. Exosomes form in a particular population of endosomes, called multivesicular bodies (MVBs), by inward budding into the lumen of the compartment. Upon fusion of MVBs with the plasma membrane, these internal vesicles are secreted. Exosomes possess a defined set of membrane and cytosolic proteins. The physiological function of exosomes is still a matter of debate, but increasing results in various experimental systems suggest their involvement in multiple biological processes. Because both cell-culture supernatants and biological fluids contain different types of lipid membranes, it is critical to perform high-quality exosome p... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817823 Sat, 01 Apr 2006 05:00:00 +0100 1817823 http://www.medworm.com/index.php?rid=1817820&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228493%26dopt%3DAbstract Authors: Peters PJ, Bos E, Griekspoor A This unit describes subcellular localization of proteins/antigens using high-resolution cryo-immunogold electron microscopy, which allows study of topological biochemistry at the ultrastructural level. This is the most sensitive procedure for immunodetection of antigens on ultrathin sections prepared from chemically fixed cells or tissues, because aldehyde fixation is the only denaturation step. The omission of harsh organic solvents (such as those used for plastic embedding) ensures better preservation of protein antigenicity. Support protocols describe how to embed fixed material in gelatin, cryosection, and mount the sections on Formvar-coated grids. This unit is accompanied by eleven videos that illustrate many of the procedures used in this ... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817820 Sat, 01 Apr 2006 05:00:00 +0100 1817820 http://www.medworm.com/index.php?rid=1817838&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228475%26dopt%3DAbstract Authors: Kornbluth S, Yang J, Powers M In this unit, Xenopus eggs are isolated from hormonally primed female frogs, and then the extract is treated with cyclohexamide so it remains in interphase of the cell cycle. In the presence of sperm chromatin and ATP, membrane vesicles in the extract fuse to assemble nuclei, making the extract suitable for studies of DNA replication and nuclear transport. PMID: 18228475 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Cell Biology) Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817838 Sun, 01 Jan 2006 05:00:00 +0100 1817838 http://www.medworm.com/index.php?rid=1817831&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228482%26dopt%3DAbstract Authors: Hu CD, Grinberg AV, Kerppola TK Protein interactions integrate stimuli from different signaling pathways and developmental programs. Bimolecular fluorescence complementation (BiFC) analysis has been developed for visualization of protein interactions in living cells. This approach is based on complementation between two fragments of a fluorescent protein when they are brought together by an interaction between proteins fused to the fragments, and it enables visualization of the subcellular locations of protein interactions in the normal cellular environment. It can be used for the analysis of many protein interactions and does not require information about the structures of the interaction partners. A multicolor BiFC approach has been developed for simultaneous visualization o... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817831 Sun, 01 Jan 2006 05:00:00 +0100 1817831 http://www.medworm.com/index.php?rid=1817830&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228483%26dopt%3DAbstract Authors: Brasaemle DL, Wolins NE Lipid droplets are organelles found in most mammalian cells, as well as various plant tissues and yeast. They are composed of a core of neutral lipids surrounded by a membrane monolayer of phospholipids and cholesterol into which specific proteins are embedded. This unit provides protocols for isolating lipid droplets from mammalian cells by discontinuous density gradient centrifugation. PMID: 18228483 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Cell Biology) Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817830 Sun, 01 Jan 2006 05:00:00 +0100 1817830 http://www.medworm.com/index.php?rid=1817829&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228484%26dopt%3DAbstract Authors: Lindstedt KA, Kovanen PT The mast cell is a multipotent inflammatory cell that has been shown to participate in the pathogenesis of a variety of diseases, such as immediate hypersensitivity reactions, arthritis, atherosclerosis, and heart failure. Upon stimulation, mast cells exocytose cytoplasmic secretory granules into their extracellular microenvironment. These granules are modified lysosomes containing preformed mediators such as histamine, neutral proteases, cytokines, and growth factors embedded in a heparin proteoglycan matrix. When exposed to the extracellular fluid, the soluble components of the granules (e.g., histamine and cytokines) diffuse away, whereas the heparin proteoglycans and the mast cell-specific neutral proteases (e.g., chymase) remain tightly bound to e... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817829 Sun, 01 Jan 2006 05:00:00 +0100 1817829 http://www.medworm.com/index.php?rid=1817828&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228485%26dopt%3DAbstract Authors: Lehmann V, Müller H, Lange BM Classical protocols for the isolation of centrosomes from higher eukaryotic cells are based on enrichment of cell organelles by density gradient centrifugation. Various successful protocols have been described that isolate centrosomes from mammalian tissue culture cells, tissue, clam oocytes, Drosophila, and yeast, to mention only some of the more frequently used sources. The material produced is subsequently used in various assays. These include functional tests such as the microtubule nucleation assay, electron microscopic study of centrosome morphology, and antigen localization; the organelles may also be used for the generation of antibodies. Furthermore, centrosomal preparations have been used for the characterization of their protein co... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817828 Sun, 01 Jan 2006 05:00:00 +0100 1817828 http://www.medworm.com/index.php?rid=1817827&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228486%26dopt%3DAbstract Authors: Rindler MJ This unit describes methods for preparing zymogen granules from rat pancreas. Zymogen granules are storage organelles in pancreatic acinar cells containing digestive enzymes that are released into the pancreatic duct. The protocols in this unit take advantage of the large size (up to 1 microm diameter) and high density (>1.20 g/cm(3) on sucrose gradients) of the granules as compared to other cellular organelles. They use a combination of differential sedimentation and density gradient separation to accomplish the purification. Similar procedures can be used to isolate zymogen granules from mouse pancreas and canine pancreas. A protocol for preparing zymogen granules from dog pancreas is also included. PMID: 18228486 [PubMed - indexed for MEDLINE] (Source: Cur... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817827 Sun, 01 Jan 2006 05:00:00 +0100 1817827 http://www.medworm.com/index.php?rid=1817826&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228487%26dopt%3DAbstract Authors: Harrison-Lowe N, Olsen LJ Peroxisomes are single-membrane-bound organelles found in virtually all eukaryotes. In plants, there are several classes of peroxisomes. Glyoxysomes are found in germinating seedlings and contain enzymes specific for the glyoxylate cycle, including isocitrate lyase and malate synthase. After seedlings become photosynthetic, leaf peroxisomes participate in reactions of the photorespiration pathway and contain characteristic enzymes such as glycolate oxidase and hydroxypyruvate reductase. As leaves begin to senesce, leaf peroxisomes are transformed back into glyoxysomes. Root peroxisomes in the nodules of legumes, for example, sequester enzymes such as allantoinase and uricase, which contribute to nitrogen metabolism in these tissues. Thus, peroxisomes ... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817826 Sun, 01 Jan 2006 05:00:00 +0100 1817826 http://www.medworm.com/index.php?rid=1817853&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228460%26dopt%3DAbstract Authors: Stark DA, Kulesa PM Selective marking of a single cell within a living embryo is often difficult due to the inaccuracy and invasiveness of standard techniques. This unit describes a minimally invasive optical protocol that uses 405-nm laser light to photoactivate a variant of green fluorescent protein (PAGFP). This method takes advantage of the accessibility of the chick embryo to inject PAGFP into a region of interest and uses electroporation to deliver the construct into cells. This unit describes in detail how single and small groups of cells (n<10) that express PAGFP can be made visually distinguishable from the host population using the photoactivation process. Included is a means to maximize the fluorescence increase due to photoactivated GFP signal and to reduce phot... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817853 Sat, 01 Oct 2005 04:00:00 +0100 1817853 http://www.medworm.com/index.php?rid=1817851&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228462%26dopt%3DAbstract Authors: Shaul Y, Seger R Mitogen-activated protein kinase (MAPK) cascades are central pathways that participate in the intracellular transmission of extracellular signals. Each of the MAPK signaling cascades seems to consist of three to five tiers of protein kinases that sequentially activate each other by phosphorylation. Since the majority of MAPK cascade components are kinases, the methods used to detect their activation involve determining phosphorylation state and protein kinase activities. The Basic Protocol describes the use of immunoblotting with specific anti-phospho antibody to detect activation of MAPK components. Alternative methods described are immunoprecipitation of desired protein kinases followed by phosphorylation of specific substrates and the use of an in-gel kinas... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817851 Sat, 01 Oct 2005 04:00:00 +0100 1817851 http://www.medworm.com/index.php?rid=1817842&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228471%26dopt%3DAbstract Authors: Denham M, Conley B, Olsson F, Cole TJ, Mollard R Stem cells are specialized cells that possess a capacity to undergo self-renewal while at the same time having the ability to give rise to at least one or more differentiated or mature cell type. They therefore represent a fundamental cornerstone during the life of all vertebrates, playing central roles in the production of new and replacement cells for tissues during development and homeostasis, including repair following disease or injury. This unit is a review of stem cells, their roles in development, and their potentials as therapeutic agents. PMID: 18228471 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Cell Biology) Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817842 Sat, 01 Oct 2005 04:00:00 +0100 1817842 http://www.medworm.com/index.php?rid=1817841&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228472%26dopt%3DAbstract Authors: Conley BJ, Denham M, Gulluyan L, Olsson F, Cole TJ, Mollard R Embryonic stem (ES) cells are pluripotent cells derived from developing mouse blastocysts in vitro that maintain long-term self renewal and the capacity to give rise to all cell types in the adult body (including some extraembryonic cell types) when subjected to the appropriate conditions. It is envisaged that the development of methods enabling controlled differentiation of mouse ES cell counterparts from human blastocysts would enable the provision of an unlimited supply of tissue for cell and tissue transplantation therapies for the repair and replacement of diseased, injured, and senescent tissue. Furthermore, derivation of mouse ES cells has allowed for the generation of thousands of gene-targeted mouse mutants... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817841 Sat, 01 Oct 2005 04:00:00 +0100 1817841 http://www.medworm.com/index.php?rid=1817854&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228459%26dopt%3DAbstract Authors: Osborne A, Flett A, Smythe E Clathrin-coated pits and vesicles represent the major ports of entry into most eukaryotic cells. As well as performing housekeeping functions (e.g., allowing cells to take up essential nutrients), the endocytic pathway participates in a number of tissue-specific events such as synaptic-vesicle recycling, control of morphogen gradients during development, downregulation of receptor tyrosine kinases, and immune surveillance. To understand the role played by clathrin-mediated uptake, it is therefore essential to have robust endocytosis assays in intact cells. The clathrin-coated vesicle cycle requires a complicated interplay of proteins and lipids that is regulated in space and time. Reconstitution assays in permeabilized cells provide a powerful appr... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817854 Fri, 01 Jul 2005 04:00:00 +0100 1817854 http://www.medworm.com/index.php?rid=1817850&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228463%26dopt%3DAbstract Authors: Schaub BE, Nair P, Rohrer J Lysosomes are terminal degradative organelles that are found in all higher eukaryotic cells. The biogenesis of lysosomes involves the transport of various acid hydrolases and transmembrane glycoproteins from their site of synthesis in the endoplasmic reticulum through the biosynthetic and endocytic pathways. Protein transport to lysosomes can be studied by a combination of techniques based on the separation of intracellular organelles. Percoll density gradient centrifugation has long been the method of choice for separating lysosomes from other organelles in cell homogenates, and accordingly, this unit describes protocols for obtaining reasonably pure lysosomal fractions from mammalian cells using Percoll density gradient separation. PMID: 18228... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817850 Fri, 01 Jul 2005 04:00:00 +0100 1817850 http://www.medworm.com/index.php?rid=1817849&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228464%26dopt%3DAbstract Authors: Bandyopadhyay D, Gatza C, Donehower LA, Medrano EE Replicative senescence, a process first described almost 40 years ago, entails irreversible growth arrest with sustained metabolic functions, and it is also associated with increased resistance to apoptotic signals. Interest in this process has increased greatly over the last 10 years, as it has been demonstrated that senescence can function as a potent tumor suppressor mechanism. Although mounting evidence suggests that the senescent phenotype is associated with an extraordinarily complex array of gene expression patterns and interactions with the microenvironment, there is only one widely accepted marker for distinguishing such cells in vitro and in vivo. This marker is the senescence-associated expression of a pH 6 beta-gal... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817849 Fri, 01 Jul 2005 04:00:00 +0100 1817849 http://www.medworm.com/index.php?rid=1817848&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228465%26dopt%3DAbstract Authors: Shockett P, Schatz D The protocols in this unit describe the transfection of adherent cells and the testing of resultant clones for inducible transactivator or target gene protein expression. Stably transfected fibroblast cell lines expressing transactivator and target gene(s) can be derived by first cotransfecting pTet-tTAk and a plasmid encoding a selectable marker and obtaining stable lines with inducible transactivator expression. These lines are subsequently stably cotransfected with plasmids encoding the target gene(s) and a second selectable marker. The procedure may also be used to cotransfect pTet-tTAk with the target gene-encoding plasmid(s) and a single selectable marker plasmid. A support protocol describes methods to test stably transfected cell lines for inducibl... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817848 Fri, 01 Jul 2005 04:00:00 +0100 1817848 http://www.medworm.com/index.php?rid=1817847&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228466%26dopt%3DAbstract Authors: Snapp E This unit describes strategies for designing functional fluorescent fusion protein constructs. Such constructs can be exploited as probes of cellular environments, protein dynamics, protein life histories, protein binding partners, and markers in living cells. The properties and uses of many currently available fluorescent proteins are discussed. In addition, alternative approaches and troubleshooting guidelines are provided. PMID: 18228466 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Cell Biology) Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817847 Fri, 01 Jul 2005 04:00:00 +0100 1817847 http://www.medworm.com/index.php?rid=1817852&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228461%26dopt%3DAbstract Authors: Hertzog M, Carlier MF A very large, ever-increasing repertoire of actin-binding proteins regulates the assembly dynamics and the spatial organization of actin filaments, thus orchestrating the motile behavior of the cell. The authors describe a series of biochemical functional assays that allow one to characterize the function of a putative actin-binding protein in actin filament dynamics. These tests allow the characterization of three types of actin-binding proteins: G-actin-sequestering proteins, profilin-like proteins, and barbed-end capping proteins. Biochemical tests include the use of sedimentation of actin filaments, polymerization assays at the barbed or pointed end of actin filaments derived from fluorescently labeled actin, thermodynamic measurements of actin assemb... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817852 Fri, 01 Apr 2005 05:00:00 +0100 1817852 http://www.medworm.com/index.php?rid=1817843&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228470%26dopt%3DAbstract Authors: Almagro S, Dimitrov S Mitotic chromosomes have fascinated scientists for several decades. Despite the numerous microscopy studies, chromosome structure is, however, still poorly understood. This is due to both the high complexity of the mitotic chromosomes and the lack of other appropriate techniques suitable for studying their organization. This unit describes a novel physical approach based on measurements of mitotic chromosome elasticity. The elasticity properties are determined by the underlying structure, and knowledge of them has allowed a description of the organization of the mitotic chromosomes and critical analysis of the available models. In this unit, a detailed protocol for the measurements of the elastic response of in vitro assembled mitotic chromosomes in Xenop... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817843 Fri, 01 Apr 2005 05:00:00 +0100 1817843 http://www.medworm.com/index.php?rid=1817840&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228473%26dopt%3DAbstract Authors: Girard M, Allaire PD, Blondeau F, McPherson PS Clathrin-coated vesicles (CCVs) are an important class of transport organelles that mediate the endocytosis of proteins and lipids at the plasma membrane and the transport of proteins from the trans-Golgi network to the endosomal/lysosomal system. The authors describe a protocol for isolating CCVs from adult rat brain using differential centrifugation, Ficoll and D(2)O-sucrose density gradient centrifugation, and velocity sedimentation in linear sucrose gradients. The application of this basic method to the isolation of CCVs from developing rat brains and to the generation of relatively crude CCVs from cultured cells is also described. Furthermore, we describe a protocol in which differential centrifugation and a series of discont... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817840 Fri, 01 Apr 2005 05:00:00 +0100 1817840 http://www.medworm.com/index.php?rid=1817839&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228474%26dopt%3DAbstract Authors: Watabe H, Kushimoto T, Valencia JC, Hearing VJ The methods used to purify early and late melanosomes are detailed. These methods include the use of highly pigmented cells to maximize recovery and the use of various sucrose density gradients to separate melanosome fractions based on their density (which is determined in large part by the amount of dense melanin pigment that they contain). Early melanosomes lacking pigment must be further purified using free-flow electrophoresis. PMID: 18228474 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Cell Biology) Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817839 Fri, 01 Apr 2005 05:00:00 +0100 1817839 http://www.medworm.com/index.php?rid=1817846&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228467%26dopt%3DAbstract Authors: Bayani J, Squire JA Comparative genomic hybridization (CGH) is a screening method based on fluorescence in situ hybridization (FISH). In contrast to conventional FISH, the metaphase target is derived from a normal peripheral blood lymphocyte culture. This target is hybridized to the test or tumor DNA, which is labeled/detected by one fluorochrome (i.e., green), and to an equal amount of labeled normal or reference DNA, which is labeled/detected by a different fluorochrome (red). It is the difference in these green/red ratios (determined by specialized software) along the length of each karyotyped chromosome that indicates the relative copy number changes in the test/tumor DNA. The basic FISH techniques reviewed in this section, the parameters for which also apply to obtaining ... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817846 Sat, 01 Jan 2005 05:00:00 +0100 1817846 http://www.medworm.com/index.php?rid=1817845&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228468%26dopt%3DAbstract Authors: Bayani J, Squire JA Sister chromatid exchange (SCE) refers to the interchange of DNA between replication products. The technique for detecting such exchanges takes advantage of the semiconservative nature of DNA synthesis. 5'-bromodeoxyuridine (BrdU) is incorporated into the newly synthesized DNA. Using standard culturing techniques, Colcemid is added to the culture and conventional cytogenetic preparations are made. Differential staining with Hoechst dye and Giemsa allows the newly synthesized DNA within a chromatid to be recognized, since BrdU incorporation results in much weaker staining. SCEs represent a point of DNA template exchange during strand synthesis, visualized as asymmetric chromatid staining or "harlequin" chromosomes. PMID: 18228468 [PubMed - indexed for ME... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817845 Sat, 01 Jan 2005 05:00:00 +0100 1817845 http://www.medworm.com/index.php?rid=1817844&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228469%26dopt%3DAbstract Authors: Bayani J, Squire JA Mitotic figures have provided important information on the mechanisms behind observed chromosomal aberrations. Variations in cell culturing and fixation methods enhance the visualization of mitotic figures. Furthermore, immunohistochemistry using a host of antibodies against protein structures involved in the cell cycle, as well as against those components involved in anchoring chromosomal structures, can reveal changes in chromosomal segregation. PMID: 18228469 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Cell Biology) Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817844 Sat, 01 Jan 2005 05:00:00 +0100 1817844 http://www.medworm.com/index.php?rid=1817875&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228438%26dopt%3DAbstract Authors: Wierzbicka-Patynowski I, Mao Y, Schwarzbauer JE The extracellular matrix acts as a framework for tissue architecture and dynamically regulates many cellular functions. Fibronectin is a ubiquitous extracellular matrix component that plays critical roles in matrix structure and in directing cell behaviors. Fibronectin is synthesized and secreted by many cell types including fibroblasts, endothelial cells, myoblasts, and astrocytes. Upon secretion, cells assemble fibronectin into a fibrillar network. During assembly, fibronectin is initially organized into fine cell-associated fibrils and, through continued accumulation of fibronectin, these fibrils are converted into a dense network of detergent-insoluble fibrils. Differential solubility in the detergent deoxycholate is the prin... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817875 Wed, 01 Dec 2004 05:00:00 +0100 1817875 http://www.medworm.com/index.php?rid=1817874&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228439%26dopt%3DAbstract Authors: Pankov R, Yamada KM This unit provides a basic protocol for non-radioactive determination of the rate of incorporation of biotinylated fibronectin into the insoluble matrix organized by cultured cells. The protocol provides quantification of changes in matrix assembly due to different experimental conditions with concomitant determinations of the activation state of various signaling molecules that may be involved in the process of matrix assembly. This unit also provides a support protocol for biotinylation of purified fibronectin. PMID: 18228439 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Cell Biology) Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817874 Wed, 01 Dec 2004 05:00:00 +0100 1817874 http://www.medworm.com/index.php?rid=1817856&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228457%26dopt%3DAbstract Authors: Craige B, Salazar G, Faundez V Synaptic vesicles are the most abundant secretory organelle in eukaryotic neural cells. Synaptic vesicles are physically distinct from other membrane-bound organelles because they are small, spherical, and highly uniform in size with a diameter of about 40 nm. Synaptic vesicles also have a characteristically low density because water and phospholipids account for 88% of an individual synaptic vesicle's weight. The homogeneous size and density of the synaptic vesicle are characteristics that are exploited in most synaptic vesicle isolation procedures. Synaptic vesicles are purified by isopycnic/velocity sedimentation and size-based purification schemes. However, protocols differ in the tissue source of vesicles, the way the tissue is homogenized, ... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817856 Wed, 01 Dec 2004 05:00:00 +0100 1817856 http://www.medworm.com/index.php?rid=1817855&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228458%26dopt%3DAbstract Authors: Esposito A, Wouters FS Fluorescent lifetime imaging microscopy is a powerful tool to enhance the contrast in images of biological samples and to investigate the local environment of a fluorochrome. FLIM allows the detection of protein-protein interactions and their biochemical state by the quantitative detection of Förster resonance energy transfer (FRET) between molecules in living cells or tissues. The availability of different spectral variants of the visible fluorescent proteins (VFPs) allows the investigation of molecular activities and protein-protein interactions in living cells by FRET as measured by FLIM. PMID: 18228458 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Cell Biology) Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817855 Wed, 01 Dec 2004 05:00:00 +0100 1817855 http://www.medworm.com/index.php?rid=1817873&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228440%26dopt%3DAbstract Authors: Le Clainche C, Carlier MF Actin-based movement can be reconstituted by using microspheres functionalized with the enzymes N-WASP or ActA, which use the Arp2/3 complex and actin to catalyze the formation of a branched actin filament network that is maintained in rapid turnover by three proteins (capping protein, profilin, and ADF). The particles continuously initiate filament assembly at their surface and are propelled, mimicking bacteria or the leading edge of motile cells. This biomimetic assay offers advantages over approaches based on living cells and cell extracts, because the physical-chemical parameters are under control. The biomimetic motility assay offers the opportunity to test the function of proteins involved in signaling pathways or actin dynamics. It is a powerfu... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817873 Fri, 01 Oct 2004 04:00:00 +0100 1817873 http://www.medworm.com/index.php?rid=1817862&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228451%26dopt%3DAbstract Authors: Dunn GA, Holt MR, Soong DY, Gray C, Zicha D Fluorescence localization after photobleaching is a new method for localized photolabeling and subsequent tracking of specific molecules within living cells. The molecular species to be located carries two different fluorophores that can be imaged independently but simultaneously by fluorescence microscopy. For the method to work, these two fluorophores should be accurately colocalized throughout the cell so that their images are closely matched. One of the fluorophores (the target fluorophore) is then rapidly photobleached at a chosen location. The unbleached (reference) fluorophore remains colocalized with the target fluorophore; thus, the subsequent fate of the photobleached molecules can be revealed by processing simultaneously a... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817862 Fri, 01 Oct 2004 04:00:00 +0100 1817862 http://www.medworm.com/index.php?rid=1817857&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228456%26dopt%3DAbstract Authors: Bayani J, Squire J Traditional FISH analysis has employed, at most, two colors of detection, a red-fluorescing fluorochrome and a green-fluorescing fluorochrome. The improvements in fluorescent imaging and development of heartier fluorochromes/dyes have enabled investigators to use several different DNA probes in one experiment. This may involve all or combinations of locus-specific probes and chromosome paints. The value of such experiments lies in the investigator obtaining far more information from one specific cell at one time, rather then carrying out separate experiments on multiple specimens prepared from the same sample, then extrapolating results. The generic term for multi-color FISH assays is M-FISH, however, the technologies behind the manner in which the fluorochr... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817857 Fri, 01 Oct 2004 04:00:00 +0100 1817857 http://www.medworm.com/index.php?rid=1817868&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228445%26dopt%3DAbstract Authors: Aparicio O, Geisberg JV, Struhl K Chromatin immunoprecipitation (ChIP) is a powerful and widely applied technique for detecting the association of individual proteins with specific genomic regions in vivo. Live cells are treated with formaldehyde to generate protein-protein and protein- DNA cross-links between molecules in close proximity on the chromatin template in vivo. DNA sequences that cross-link with a given protein are selectively enriched and reversal of the formaldehyde cross-link permits recovery and quantitative analysis of the immunoprecipitated DNA. As formaldehyde inactivates cellular enzymes essentially immediately upon addition to cells, ChIP provides snapshots of protein-protein and protein- DNA interactions at a particular time point, and hence is useful for... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817868 Wed, 01 Sep 2004 04:00:00 +0100 1817868 http://www.medworm.com/index.php?rid=1817867&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228446%26dopt%3DAbstract Authors: Velázquez-Campoy A, Ohtaka H, Nezami A, Muzammil S, Freire E In the last two decades, isothermal titration calorimetry (ITC) has become the preferred technique to determine the binding energetics of biological processes, including protein-ligand binding, protein-protein binding, DNA-protein binding, protein-carbohydrate binding, protein-lipid binding, and antigen-antibody binding. In this unit several protocols are presented, ranging from the basic ones that are aimed at characterizing binding of moderate affinity to advanced protocols that are aimed at determining very high or very low affinity binding processes. Also, alternate protocols for special cases (homodimeric proteins and unstable proteins) and additional information accessible by ITC (heat capacity and protona... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817867 Wed, 01 Sep 2004 04:00:00 +0100 1817867 http://www.medworm.com/index.php?rid=1817861&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228452%26dopt%3DAbstract Authors: Bayani J, Squire JA This unit provides a brief overview of cytogenetic analysis of interphase ansd metaphase chromosomes. It includes a description of the methods provided in the units that follow. PMID: 18228452 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Cell Biology) Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817861 Wed, 01 Sep 2004 04:00:00 +0100 1817861 http://www.medworm.com/index.php?rid=1817860&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228453%26dopt%3DAbstract Authors: Bayani J, Squire JA Cytogenetic specimens are prepared from short-term primary cultures established from tissue samples or from established cell lines. Cultures that are (80% confluent are treated with Colcemid to enrich for cells in mitosis. Cells are then swollen hypotonically and fixed before slides are prepared. Preparing good metaphase spreads is technically challenging, This unit provides a good discussion of the factors that go into making a good preparation. PMID: 18228453 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Cell Biology) Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817860 Wed, 01 Sep 2004 04:00:00 +0100 1817860 http://www.medworm.com/index.php?rid=1817859&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228454%26dopt%3DAbstract Authors: Bayani J, Squire JA Traditional banding of metaphase chromosomes allows identification of individual chromosomes and detection of gross chromosomal anomalies and abnormal chromosome structures. Giemsa (G) banding is the older and more familiar nonfluorescent technique that produces characteristic banding patterns. DAPI , a fluorescent DNA stain, produces banding patterns similar to those of G-banding and is much simpler to perform. PMID: 18228454 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Cell Biology) Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817859 Wed, 01 Sep 2004 04:00:00 +0100 1817859 http://www.medworm.com/index.php?rid=1817858&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228455%26dopt%3DAbstract Authors: Bayani J, Squire JA Fluorescence in situ Hybridization (FISH) involves the preparation of two main components: the DNA probe and the target DNA to which the probe will be hybridized. The DNA probe typically comes from cloned sources such as plasmids, cosmids, PACs, YACs, or BACs; where the insert may contain a specific gene or originate from a specific chromosomal locus. Whole-chromosome paints may also be used but are usually applicable to metaphase preparations. The purified DNA can then be labeled and detected indirectly using haptens, or labeled directly using fluorochrome or dye-conjugated nucleotides. Labeling strategies are also variable, employing standard nick translation or PCR labeling methods. The target DNA can take the form of chromosomes spreads or interphase nu... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817858 Wed, 01 Sep 2004 04:00:00 +0100 1817858 http://www.medworm.com/index.php?rid=1817872&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228441%26dopt%3DAbstract Authors: Pankov R This unit provides a basic protocol for assaying the level of activity, as well as the activation capacity and dynamics of inhibition of Akt/PKB in cultured cells. All these data can be obtained in a single nonradioactive experiment by standard techniques including immunoblotting and immunodetection with phosphospecific antibodies.This unit also provides a support protocol for assaying the membrane translocation of Akt. PMID: 18228441 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Cell Biology) Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817872 Sat, 01 May 2004 04:00:00 +0100 1817872 http://www.medworm.com/index.php?rid=1817871&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228442%26dopt%3DAbstract Authors: Chow CW, Downey GP, Grinstein S Phagocytosis is an essential component of the innate immune response. Invading microorganisms are engulfed by neutrophils and macrophages, and subsequently killed by a complex series of reactions that require fusion of the phagocytic vacuole with multiple endomembrane organelles. This vacuolar remodeling process, known as phagosomal maturation, is often the target of intracellular parasites that evade killing by the host immune system. This unit describes detailed protocols for the assessment of phagosome formation and maturation. Particular attention is given to Fc- and complement-receptor mediated phagocytosis, but the protocols provided can be readily adapted to other types of phagocytic processes. PMID: 18228442 [PubMed - indexed for MED... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817871 Sat, 01 May 2004 04:00:00 +0100 1817871 http://www.medworm.com/index.php?rid=1817870&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228443%26dopt%3DAbstract Authors: Brymora A, Valova VA, Robinson PJ The aim of this unit is to provide a method for the identification of new protein-protein interactions. Pull-down experiments with GST fusion proteins attached to glutathione beads are a screening technique for identification of protein-protein interactions. When coupled with mass spectrometry, pull-downs can be considered as the protein-based equivalent of a yeast two-hybrid screen. To improve the isolation of specific binding partners, pull-down methods are described involving the use of cross-linking, large-scale tissue lysates, and spin columns. Alternative techniques are detailed for isolating activation state-dependent protein interactions with small GTPases. Appropriate methods of sample preparation for mass spectrometry-based identific... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817870 Sat, 01 May 2004 04:00:00 +0100 1817870 http://www.medworm.com/index.php?rid=1817869&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228444%26dopt%3DAbstract Authors: Schuck P, Boyd LF, Andersen PS In recent years, optical evanescent wave biosensors have been used to characterize protein-protein interactions, including determination of equilibrium binding constants and bimolecular rate constants. This surface binding technique can provide information about chemical on-rate constants, the lifetimes of complexes formed, and the time course of the signal. This unit provides a thorough, well-illustrated discussion of the principles of optical biosensors, experimental design, ligand immobilization, experimental data analysis, and common obstacles and possible solutions. PMID: 18228444 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Cell Biology) Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817869 Sat, 01 May 2004 04:00:00 +0100 1817869 http://www.medworm.com/index.php?rid=1817866&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228447%26dopt%3DAbstract Authors: Haince JF, Poirier GG, Kirkland JB Poly(ADP-ribosyl)ation is a post-translational modification catalyzed mostly by the 116-kDa enzyme poly(ADP-ribose) polymerase-1 (PARP-1), a nuclear enzyme that transfers an ADP-ribose moiety onto a limited number of nuclear proteins, including itself. When cells are exposed to environmental stresses such as alkylating agents or free radicals, there is up to a 500-fold increase in net poly(ADP-ribose) synthesis in response to DNA strand breaks. The enzyme responsible for 80% to 90% of this stimulated poly(ADP-ribose) synthesis is PARP-1, while other PARPs are responsible for the remaining 10% to 20%. The physiological meaning of these phenomena is not clear; however, it can be interpreted as a way of translating an event occurring on DNA to t... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817866 Sun, 01 Feb 2004 05:00:00 +0100 1817866 http://www.medworm.com/index.php?rid=1817865&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228448%26dopt%3DAbstract Authors: Pozarowski P, Grabarek J, Darzynkiewicz Z Common methods applicable to flow cytometry make it possible to: (1) identify and quantify dead or dying cells, (2) reveal a mode of cell death (apoptosis or necrosis), and (3) study mechanisms involved in cell death. Gross changes in cell morphology and chromatin condensation, which occur during apoptosis, can be detected by analysis with laser light beam scattering. Early events of apoptosis, dissipation of the mitochondrial transmembrane potential and caspase activation, can be detected using either fluorochrome reporter groups or appropriate antibodies. Exposure of phosphatidylserine on the exterior surface of the plasma membrane can be detected by the binding of fluoresceinated annexin V. Another apoptotic event, DNA fragmentation... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817865 Sun, 01 Feb 2004 05:00:00 +0100 1817865 http://www.medworm.com/index.php?rid=1817864&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228449%26dopt%3DAbstract Authors: Wilson SM, Chambers AF Experimental metastasis assays are used to measure the ability of cancer cells to grow in secondary organs following injection of the cells into the circulation of an experimental animal. The chicken embryo provides an alternative to the more commonly used assays in mice. Details of the experimental metastasis assay in chick embryo are provided, including protocols for maintenance of fertilized chicken eggs, injection of cells into the circulation of 11-day-old chick embryos, recovery and quantification of cancer cells from chick liver using the ouabain plating assay, labeling of cells with fluorescent nanospheres, and monitoring of individual steps in the metastatic process in the chick chorioallantoic membrane using intravital videomicroscopy. These as... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817864 Sun, 01 Feb 2004 05:00:00 +0100 1817864 http://www.medworm.com/index.php?rid=1817863&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228450%26dopt%3DAbstract Authors: Yamada SS Cultured keratinocytes have been used by a number of investigators in studies investigating wound repair and carcinogenesis, and they have also proven useful as a model for differentiation. This unit describes a protocol for establishing human keratinocytes in tissue culture. Human newborn foreskins are proteolytically digested to separate the epidermis and the dermis, and keratinocytes are obtained from the epidermis. PMID: 18228450 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Cell Biology) Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817863 Sun, 01 Feb 2004 05:00:00 +0100 1817863 http://www.medworm.com/index.php?rid=1817880&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228433%26dopt%3DAbstract Authors: Rocheleau JV, Piston DW Two-photon excitation microscopy is an alternative to confocal microscopy that provides advantages in three-dimensional and deep tissue imaging. This unit will describe the basic physical principles of two-photon excitation and discuss the advantages and limitations of its use in laser-scanning microscopy. The advantages of two-photon microscopy are reduced phototoxicity, increased imaging depth, and the ability to initiate localized photochemistry. PMID: 18228433 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Cell Biology) Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817880 Sat, 01 Nov 2003 05:00:00 +0100 1817880 http://www.medworm.com/index.php?rid=1817879&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228434%26dopt%3DAbstract Authors: Jaiswal JK, Simon SM The wavelength of light imposes a physical limit of approximately 400 nm on the maximum resolution that can be achieved using light microscopy. This unit will describe the use of total internal reflection fluorescence microscopy (TIR-FM), or evanescent wave microscopy, an approach that partially overcomes this physical limit and permits one to selectively image just those fluorophores in the optical plane (along the z axis) within 50 nm of the cell surface. TIR-FM works by means of limiting the depth of penetration of the excitation light within this narrow region. This narrow excitatory plane not only provides a high signal-to-noise ratio but also minimizes the photodamage to the cell. PMID: 18228434 [PubMed - indexed for MEDLINE] (Source: Current Pro... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817879 Sat, 01 Nov 2003 05:00:00 +0100 1817879 http://www.medworm.com/index.php?rid=1817878&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228435%26dopt%3DAbstract Authors: Baggett JJ, Shaw JD, Sciambi CJ, Watson HA, Wendland B This unit describes the use of several different fluorescence methods for labeling yeast cells. It includes methods to label the vacuole, the actin cytoskeleton, and chitin deposits on cell walls (bud scars), as well as methods for visualizing specific proteins in live cells with GFP chimeras and in fixed cells by immunofluorescence. PMID: 18228435 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Cell Biology) Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817878 Sat, 01 Nov 2003 05:00:00 +0100 1817878 http://www.medworm.com/index.php?rid=1817884&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228429%26dopt%3DAbstract Authors: Potter H Electroporation-the use of high-voltage electric shocks to introduce DNA into cells-can be used with most cell types, yields a high frequency of both stable transformation and transient gene expression, and, because it requires fewer steps, can be easier than alternate techniques. In this unit, the basic protocol describes the electroporation of mammalian cells and an alternate protocol outlines modifications for preparation and transfection of plant protoplasts. PMID: 18228429 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Cell Biology) Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817884 Fri, 01 Aug 2003 04:00:00 +0100 1817884 http://www.medworm.com/index.php?rid=1817883&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228430%26dopt%3DAbstract Authors: Hawley-Nelson P, Ciccarone V The development of high-efficiency methods for the introduction of functional genetic material into eukaryotic cells using cationic lipids has accelerated biological research in the studies of gene expression, control of cell growth, and cell lineage. Transfection mediated by cationic lipids is commonly used in industrial protein production as well as in some clinical gene therapy protocols. Replacing our previous unit on this topic, this new version describes how to perform transfection of adherent and suspension cells, insect cells, and RNA transfection using the cationic lipid system. PMID: 18228430 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Cell Biology) Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817883 Fri, 01 Aug 2003 04:00:00 +0100 1817883 http://www.medworm.com/index.php?rid=1817882&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228431%26dopt%3DAbstract Authors: Rose JK When embarking upon any transfection procedure, a critical first step is to optimize conditions. Every mammalian cell type has a characteristic set of requirements for optimal introduction of foreign DNA; there is a tremendous degree of variability in the transfection conditions that work, even among cell types that are very similar to one another. Often, an experimenter must screen a wide variety of cell types for a desired regulatory trait, such as an appropriate response to a particular effector molecule. It is thus helpful to have a straightforward, systematic approach to optimizing transfection efficiency. This unit provides guidelines to optimizing transfection experiments. PMID: 18228431 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Cell Biolo... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817882 Fri, 01 Aug 2003 04:00:00 +0100 1817882 http://www.medworm.com/index.php?rid=1817881&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228432%26dopt%3DAbstract Authors: Snapp EL, Altan N, Lippincott-Schwartz J This unit describes fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) using commercially available confocal scanning laser microscopy (CSLM). Photobleaching is the photo-induced change in a fluorphore that abolishes that molecule's fluorescence. The different characteristics of green fluorescent protein (GFP) chimeras in a cell can be studied by FRAP, in which a selected region of the cell is photobleached with intense light. The movement of unbleached molecules into a photobleached region is quantified by imaging with an attenuated light source. The movement of molecules between cellular compartments can be determined by FLIP, in which the same region of a cell expressing a GFP chimera is ... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817881 Fri, 01 Aug 2003 04:00:00 +0100 1817881 http://www.medworm.com/index.php?rid=1817876&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228437%26dopt%3DAbstract Authors: Kucik DF This unit describes the analysis of dynamic cell adhesion using a flow chamber assay. The flow chamber enables the researcher to reconstruct cell systems in the presence of shear stress to assay adhesion under well&defined forces. These assays are most commonly used to study leukocyte adhesion, either to cultured endothelial cell monolayers or to purified substrates, simulating physiological interactions of leukocytes with endothelial cells. This assay can be also be used to characterize transient adhesive events or adhesion strengthening even for cells that do not normally experience shear stress, because contact time between cells and substrates and anti&adhesive forces can be closely regulated by stopping and starting the flow. Flow chamber assays are also ... Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817876 Thu, 01 May 2003 04:00:00 +0100 1817876 http://www.medworm.com/index.php?rid=1817877&cid=s_38097_171_f&fid=38097&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D18228436%26dopt%3DAbstract Authors: Delahunty C, Yates JR Liquid chromatography techniques have been successfully coupled with mass spectrometers to provide a robust method for the identification of proteins in mixtures. Chromatography can be performed in-line with the mass spectrometer and data acquisition can be directly interfaced with search algorithms for identification by correlation with databases. PMID: 18228436 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Cell Biology) Current Protocols in Cell Biology journals http://www.medworm.com/rss/comments.php?id=1817877 Sat, 01 Feb 2003 05:00:00 +0100 1817877