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Overview: generation of gene knockout mice.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 totipot...
Source: Current Protocols in Cell Biology - August 31, 2009 Category: Cytology Authors: Hall B, Limaye A, Kulkarni AB Tags: Curr Protoc Cell Biol Source Type: journals

Manipulation of mouse embryonic stem cells for knockout mouse production.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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: 19731...
Source: Current Protocols in Cell Biology - August 31, 2009 Category: Cytology Authors: Limaye A, Hall B, Kulkarni AB Tags: Curr Protoc Cell Biol Source Type: journals

Generation of gene knockout mice by ES cell microinjection.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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...
Source: Current Protocols in Cell Biology - August 31, 2009 Category: Cytology Authors: Longenecker G, Kulkarni AB Tags: Curr Protoc Cell Biol Source Type: journals

Isolation and biochemical characterization of amyloid plaques and paired helical filaments.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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...
Source: Current Protocols in Cell Biology - August 31, 2009 Category: Cytology Authors: Rostagno A, Ghiso J Tags: Curr Protoc Cell Biol Source Type: journals

In vitro GEF and GAP assays.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 num...
Source: Current Protocols in Cell Biology - May 31, 2009 Category: Cytology Authors: Eberth A, Ahmadian MR Tags: Curr Protoc Cell Biol Source Type: journals

Methods used to study respiratory virus infection.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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...
Source: Current Protocols in Cell Biology - May 31, 2009 Category: Cytology Authors: Flaño E, Jewell NA, Durbin RK, Durbin JE Tags: Curr Protoc Cell Biol Source Type: journals

Compartmented neuron cultures for directional infection by alpha herpesviruses.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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)
Source: Current Protocols in Cell Biology - May 31, 2009 Category: Cytology Authors: Curanović D, Ch'ng TH, Szpara M, Enquist L Tags: Curr Protoc Cell Biol Source Type: journals

HIV-1 interactions with cells: from viral binding to cell-cell transmission.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 tran...
Source: Current Protocols in Cell Biology - May 31, 2009 Category: Cytology Authors: Janas AM, Wu L Tags: Curr Protoc Cell Biol Source Type: journals

Culturing MDCK cells in three dimensions for analyzing intracellular dynamics.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 fo...
Source: Current Protocols in Cell Biology - May 31, 2009 Category: Cytology Authors: Elia N, Lippincott-Schwartz J Tags: Curr Protoc Cell Biol Source Type: journals

Measurement of adhesion under flow conditions.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 ...
Source: Current Protocols in Cell Biology - May 31, 2009 Category: Cytology Authors: Kucik DF Tags: Curr Protoc Cell Biol Source Type: journals

Overview: engineering transgenic constructs and mice.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 ba...
Source: Current Protocols in Cell Biology - March 1, 2009 Category: Cytology Authors: Haruyama N, Cho A, Kulkarni AB Tags: Curr Protoc Cell Biol Source Type: journals

Generation of transgenic mice.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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)
Source: Current Protocols in Cell Biology - March 1, 2009 Category: Cytology Authors: Cho A, Haruyama N, Kulkarni AB Tags: Curr Protoc Cell Biol Source Type: journals

Visualization of cellular phosphoinositide pools with GFP-fused protein-domains.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 va...
Source: Current Protocols in Cell Biology - March 1, 2009 Category: Cytology Authors: Balla T, Várnai P Tags: Curr Protoc Cell Biol Source Type: journals

Fabrication and application of nanofibrous scaffolds in tissue engineering.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 (Bas...
Source: Current Protocols in Cell Biology - March 1, 2009 Category: Cytology Authors: Li WJ, Tuan RS Tags: Curr Protoc Cell Biol Source Type: journals

BK virus (BKV): infection, propagation, quantitation, purification, labeling, and analysis of cell entry.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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, det...
Source: Current Protocols in Cell Biology - March 1, 2009 Category: Cytology Authors: Moriyama T, Sorokin A Tags: Curr Protoc Cell Biol Source Type: journals

Isolation of dense core secretory vesicles from pancreatic endocrine cells by differential and density gradient centrifugation.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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)
Source: Current Protocols in Cell Biology - March 1, 2009 Category: Cytology Authors: Hutton JC, Wong R, Davidson HW Tags: Curr Protoc Cell Biol Source Type: journals

Embryonic organ culture.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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)
Source: Current Protocols in Cell Biology - December 1, 2008 Category: Cytology Authors: Sakai T, Onodera T Tags: Curr Protoc Cell Biol Source Type: journals

Three-Dimensional Tissue Models of Normal and Diseased Skin.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 ...
Source: Current Protocols in Cell Biology - December 1, 2008 Category: Cytology Authors: Carlson MW, Alt-Holland A, Egles C, Garlick JA Tags: Curr Protoc Cell Biol Source Type: journals

Monitoring mRNA export.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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...
Source: Current Protocols in Cell Biology - December 1, 2008 Category: Cytology Authors: Tokunaga K, Tani T Tags: Curr Protoc Cell Biol Source Type: journals

Isolation of neuromelanin granules.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 i...
Source: Current Protocols in Cell Biology - December 1, 2008 Category: Cytology Authors: Tribl F Tags: Curr Protoc Cell Biol Source Type: journals

Photoactivated Localization Microscopy (PALM) of Adhesion Complexes.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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...
Source: Current Protocols in Cell Biology - December 1, 2008 Category: Cytology Authors: Shroff H, White H, Betzig E Tags: Curr Protoc Cell Biol Source Type: journals

Use of hyaluronan-derived hydrogels for three-dimensional cell culture and tumor xenografts.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 hyalu...
Source: Current Protocols in Cell Biology - September 1, 2008 Category: Cytology Authors: Serban MA, Scott A, Prestwich GD Tags: Curr Protoc Cell Biol Source Type: journals

Matrix metalloproteinases.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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)
Source: Current Protocols in Cell Biology - September 1, 2008 Category: Cytology Authors: Birkedal-Hansen H, Yamada S, Windsor J, Pollard AH, Lyons G, Stetler-Stevenson W, Birkedal-Hansen B Tags: Curr Protoc Cell Biol Source Type: journals

Analysis of endocytic trafficking by single-cell fluorescence ratio imaging.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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...
Source: Current Protocols in Cell Biology - September 1, 2008 Category: Cytology Authors: Barriere H, Lukacs GL Tags: Curr Protoc Cell Biol Source Type: journals

Making giant unilamellar vesicles via hydration of a lipid film.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 ...
Source: Current Protocols in Cell Biology - September 1, 2008 Category: Cytology Authors: Manley S, Gordon VD Tags: Curr Protoc Cell Biol Source Type: journals

Purification of intact chloroplasts from Arabidopsis and spinach leaves by isopycnic centrifugation.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 smal...
Source: Current Protocols in Cell Biology - September 1, 2008 Category: Cytology Authors: Seigneurin-Berny D, Salvi D, Joyard J, Rolland N Tags: Curr Protoc Cell Biol Source Type: journals

Assays for ribosomal RNA processing and ribosome assembly.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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...
Source: Current Protocols in Cell Biology - June 1, 2008 Category: Cytology Authors: Pestov DG, Lapik YR, Lau LF Tags: Curr Protoc Cell Biol Source Type: journals

Visualization and measurement of DNA methyltransferase activity in living cells.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 t...
Source: Current Protocols in Cell Biology - June 1, 2008 Category: Cytology Authors: Schermelleh L, Spada F, Leonhardt H Tags: Curr Protoc Cell Biol Source Type: journals

Isolation of microtubules and microtubule proteins.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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-dimensio...
Source: Current Protocols in Cell Biology - June 1, 2008 Category: Cytology Authors: Avila J, Soares H, Fanarraga ML, Zabala JC Tags: Curr Protoc Cell Biol Source Type: journals

Fluorescence imaging techniques for studying Drosophila embryo development.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 organ...
Source: Current Protocols in Cell Biology - June 1, 2008 Category: Cytology Authors: Mavrakis M, Rikhy R, Lilly M, Lippincott-Schwartz J Tags: Curr Protoc Cell Biol Source Type: journals

Quantitative colocalization analysis of confocal fluorescence microscopy images.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 calculation...
Source: Current Protocols in Cell Biology - June 1, 2008 Category: Cytology Authors: Zinchuk V, Zinchuk O Tags: Curr Protoc Cell Biol Source Type: journals

Visualizing protease activity in living cells: from two dimensions to four dimensions.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 cl...
Source: Current Protocols in Cell Biology - June 1, 2008 Category: Cytology Authors: Jedeszko C, Sameni M, Olive MB, Moin K, Sloane BF Tags: Curr Protoc Cell Biol Source Type: journals

Rho GTPase activation assays.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 succe...
Source: Current Protocols in Cell Biology - March 1, 2008 Category: Cytology Authors: Pellegrin S, Mellor H Tags: Curr Protoc Cell Biol Source Type: journals

Photoactivation and imaging of photoactivatable fluorescent proteins.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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)
Source: Current Protocols in Cell Biology - March 1, 2008 Category: Cytology Authors: Patterson GH Tags: Curr Protoc Cell Biol Source Type: journals

Isolation of amyloplasts.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 - index...
Source: Current Protocols in Cell Biology - March 1, 2008 Category: Cytology Authors: Denyer K, Pike M Tags: Curr Protoc Cell Biol Source Type: journals

Two-dimensional blue native polyacrylamide gel electrophoresis.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 ...
Source: Current Protocols in Cell Biology - March 1, 2008 Category: Cytology Authors: Schamel WW Tags: Curr Protoc Cell Biol Source Type: journals

Visualization of RNA using fluorescence complementation triggered by aptamer-protein interactions (RFAP) in live bacterial cells.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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)
Source: Current Protocols in Cell Biology - December 1, 2007 Category: Cytology Authors: Valencia-Burton M, Broude NE Tags: Curr Protoc Cell Biol Source Type: journals

Production of papillomavirus-based gene transfer vectors.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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...
Source: Current Protocols in Cell Biology - December 1, 2007 Category: Cytology Authors: Buck CB, Thompson CD Tags: Curr Protoc Cell Biol Source Type: journals

Isolation of endoplasmic reticulum, mitochondria, and mitochondria-associated membrane fractions from transfected cells and from human cytomegalovirus-infected primary fibroblasts.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 transf...
Source: Current Protocols in Cell Biology - December 1, 2007 Category: Cytology Authors: Bozidis P, Williamson CD, Colberg-Poley AM Tags: Curr Protoc Cell Biol Source Type: journals

Scanning electron microscopy of cell surface morphology.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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: 18228...
Source: Current Protocols in Cell Biology - December 1, 2007 Category: Cytology Authors: Passey S, Pellegrin S, Mellor H Tags: Curr Protoc Cell Biol Source Type: journals

One-dimensional SDS gel electrophoresis of proteins.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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, ...
Source: Current Protocols in Cell Biology - December 1, 2007 Category: Cytology Authors: Gallagher SR Tags: Curr Protoc Cell Biol Source Type: journals

Basic techniques in mammalian cell tissue culture.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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)
Source: Current Protocols in Cell Biology - September 1, 2007 Category: Cytology Authors: Phelan MC Tags: Curr Protoc Cell Biol Source Type: journals

Analysis of regulated secretion using PC12 cells.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 secret...
Source: Current Protocols in Cell Biology - September 1, 2007 Category: Cytology Authors: Taupenot L Tags: Curr Protoc Cell Biol Source Type: journals

The fluorescent protein color palette.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 prob...
Source: Current Protocols in Cell Biology - September 1, 2007 Category: Cytology Authors: Olenych SG, Claxton NS, Ottenberg GK, Davidson MW Tags: Curr Protoc Cell Biol Source Type: journals

Hematoendothelial differentiation of human embryonic stem cells.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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...
Source: Current Protocols in Cell Biology - September 1, 2007 Category: Cytology Authors: Vodyanik MA, Slukvin II Tags: Curr Protoc Cell Biol Source Type: journals

Neural differentiation of human ES cells.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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. T...
Source: Current Protocols in Cell Biology - September 1, 2007 Category: Cytology Authors: Cohen MA, Itsykson P, Reubinoff BE Tags: Curr Protoc Cell Biol Source Type: journals

In vivo imaging using quantum-dot-conjugated probes.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 too...
Source: Current Protocols in Cell Biology - September 1, 2007 Category: Cytology Authors: S Lidke D, Nagy P, J Arndt-Jovin D Tags: Curr Protoc Cell Biol Source Type: journals

Analyzing real-time video microscopy: the dynamics and geometry of vesicles and tubules in endocytosis.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 abo...
Source: Current Protocols in Cell Biology - June 1, 2007 Category: Cytology Authors: Hamilton N, Kerr MC, Burrage K, Teasdale RD Tags: Curr Protoc Cell Biol Source Type: journals

Imaging tumor cell movement in vivo.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 ...
Source: Current Protocols in Cell Biology - June 1, 2007 Category: Cytology Authors: Kedrin D, Wyckoff J, Sahai E, Condeelis J, Segall JE Tags: Curr Protoc Cell Biol Source Type: journals

Replication labeling with halogenated thymidine analogs.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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...
Source: Current Protocols in Cell Biology - June 1, 2007 Category: Cytology Authors: Yokochi T, Gilbert DM Tags: Curr Protoc Cell Biol Source Type: journals