Apoptosis and Cell Proliferation (522915), страница 23
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During this period, MTT is converted into a colored, water-insoluble formazan saltby the metabolic activity of viable cells.P The insoluble formazan is solubilized.P The amount of formazan is quantified by an ELISA plate reader at 550–600 nm.Cell Proliferation Kit I (MTT)LimitationsFor productinformation, seepage 73of this guidehigh numbersXTT Assay62P XTT solution is added to cells cultured in MTP and the cells are incubated (usually forNon-isotopic2–4 h). During this period, XTT is converted into a colored, soluble formazan salt bythe metabolic activity of viable cells.P The amount of formazan is quantified by an ELISA plate reader at 450–500 nm.Cell Proliferation Kit II (XTT)WST-1 AssayP WST-1 solution is added to cells cultured in MTP and the cells are incubated (usuallyNon-isotopicfor 0.5–2 h).
During this period, WST-1 is converted into a colored, soluble formazansalt by the metabolic activity of viable cells.P The amount of formazan is quantified by an ELISA plate reader at 420–480 nm.Cell Proliferation Reagent (WST-1)P No transfer of the cells; the entire assay is performed in a single MTP P Assay is not linear over a broad logarithmic range of cell proliferationdue to the ELISA plate readerP Soluble reaction productP Can take multiple time points in a single assayP XTT working solution has to be prepared shortly before useP XTT is not metabolized by all cell typespage 74of this guideP No transfer of the cells; the entire assay is performed in a single MTP P Assay is not linear over a broad logarithmic range of cell proliferationdue to the ELISA plate readerP Soluble reaction productP Repeated measurement of the assayP WST-1 is not metabolized by all cell typesP Ready-to-use solutionpage 75of this guideG Table 17: Summary of methods to study metabolic activity in cell populations.2.2.1.4 Single reagents for the measurement of DNA synthesisF Table 18: Single reagents availablefor detection of DNA fragmentation.ProductCat.
No.Pack SizeFixDenat1 758 7644 x 100 ml (2000 tests)Anti-Bromodeoxyuridine-AP, F(ab’)2 fragments1 758 74850 µg (500 µl)Anti-Bromodeoxyuridine-Peroxidase, Fab fragments,formalin grade1 585 86015 U85Cell Proliferation and ViabilityMethod/Roche Molecular Biochemicals product2Methods for studying cell proliferation and viability in individual cellsAssays that measure DNA synthesis2.2.2 Methods for studying cellproliferation and viability in individualcellsAs mentioned in Section 2.1, the viability aswell as proliferation of individual cells canbe assessed by standard microscopic methods.
For instance, cells may be treated witha vital stain or exclusion dye and counteddirectly in a hemocytometer. The same cellparameters may be determined by flow cytometry if the cells are differentially stainedwith fluorescent dyes that bind DNA(DNA fluorochromes), see also section1.2.2.3 on page 36 of this guide.Cell Proliferation and ViabilityIn the following sections we will describedetails of the following proliferation assays:P Assays that measure DNA synthesis:As outlined above, if labeled DNA precursors are added to the cell culture,cells that are about to divide incorporate this precursor into their DNA (described on the following pages of thisguide)P Asssays that monitor expression of cellcycle-associated antigens: Moleculesthat regulate the cell cycle are measuredeither by their activity (e.g.
CDK kinaseassays) or by quantifying their amounts(e.g. Western blots, ELISA, or immunohistochemistry) (described on page 97of this guide).2In the following sections we will describedetails of several of these proliferation assays.2.2.2.1 Assays that measure DNAsynthesisStudies of cell proliferation in vivo as wellas on individual cells in vitro frequentlyemploy [3H]-TdR to label the DNA of replicating cells and autoradiography to revealthe radioactive label. As a nonradioactivealternative, bromodeoxyuridine (BrdU)can be used to label proliferating cellsin vivo and in vitro. Incorporated BrdUcan be detected by immunohistochemistry, immunocytochemistry or flow cytometry73, 74.Immunochemical techniques allow boththe visualization of dividing cells and thedetection of tissue morphology by counterstaining (e.g., with hematoxylin and/oreosin).
Thus, it is possible to visualize cellswhich have incorporated BrdU into DNAin its natural environment and to localizecell position in the tissue.As only those cells which are actually in theS-phase (DNA-synthesis) of the cell cyclewill be labeled, the so-called “labeling index”75 can be determined if the labeled nucleotide ([3H]-TdR or BrdU) is present foronly short periods of time (e.g. 15–60 minutes). The “labeling index” (proportion ofS-phase cells in an asynchronously growing population) is calculated by dividingthe number of labeled cells by the totalnumber of cells in the entire population.While short labeling periods (pulse labeling) are suitable to quantify the percentage of S-phase cells within a cellular population, longer labeling periods (e.g. for awhole cell cycle transition) can be used todetermine a replicating population.Roche Molecular Biochemicals offers several kits and reagents for measuring proliferating cells by BrdU incorporation.These products are described on the following pages.86Methods for studying cell proliferation and viability in individual cellsAssays that measure DNA synthesisBrdU Labeling and Detection Kit ICat.
No. 1 296 736100 testsBrdU Labeling and Detection Kit II100 testsType1st generation immunostaining assays for fluorescence (Kit I) or light (Kit II)microscopyUseful forDetection of BrdU-labeled DNA in proliferating individual cellsSamplesCultured or freshly isolated cells, tissue explants or sectionsMethodIncubation of cells with BrdU, or injection into an animal, followed by nuclease digestion of DNA of cells or tissue sections and indirect immunodetection(with anti-BrdU and a secondary antibody) of incorporated BrdU labelTimeapprox.
2–3 h (+ BrdU labeling)Significance of kits: The BrdU Labelingand Detection Kits I and II offer an indirectimmunostaining method for visualizingproliferating cells under a fluorescencemicroscope (Kit I) or under a light microscope (Kit II). The kits detect BrdU-labeledDNA with an anti-BrdU antibody, thenmake the antibody-labeled DNA visiblewith either a fluorescein-labeled (Kit I)or an alkaline phosphatase-labeled antimouse secondary antibody (Kit II).Cell Proliferation and ViabilityCat.
No. 1 299 964Other applications: For examples of howthe BrdU Labeling and Detection Kits Iand II can be used in the laboratory, seeAppendix, page 125.2Note: These kits belong to the first generation of kits used to measure DNA synthesis.The same assay procedure has been optimized and improved in the second generation of kits, namely the In Situ Cell Proliferation Kit, FLUOS (for flow cytometryand fluorescence microscopy) and the InSitu Cell Proliferation Kit, AP (for lightmicroscopy). For a detailed description ofthese second generation kits, see the following pages.87Methods for studying cell proliferation and viability in individual cellsAssays that measure DNA synthesisIn Situ Cell Proliferation Kit, FLUOSCell Proliferation and ViabilityCat.
No. 1 810 740100 testsTypeDirect immuno-fluorescence staining for flow cytometry or fluorescencemicroscopyUseful forDetection of BrdU-labeled DNA in proliferating individual cellsSamplesCultured or freshly isolated cells, tissue explants or sectionsMethodIncubation of cells with BrdU, or injection of BrdU into an animal followedby denaturation of DNA of cells or tissue sections and direct immunodetection of incorporated BrdU labelTimeapprox. 2 h (+ 0.5–4 h BrdU labeling)Significance of kit: Bromodeoxyuridine(BrdU) is only incorporated into the DNAof proliferating cells. Short periods (15–60min) of incubation in vitro with BrdU willtag only cells actually going through the Sphase of the cell cycle.
Alternatively, BrdUcan be injected into an animal to label growing cells in vivo. The In Situ Cell Proliferation Kit, FLUOS can detect proliferatingcells in culture or in tissues which havebeen tagged by in vitro or in vivo BrdUlabeling. Analysis can be done by flow cytometry or by fluorescence microscopy.Test principle: The BrdU solution andfluorescein-conjugated anti-BrdU antibody supplied in the kit allow BrdU labeling and detection of proliferating cells.The procedure (Figure 60 and Flow Charts20–22) involves:21A: Incubating growing animal tissue orcells in vitro with BrdUorFigure 60: How the In SituCell Proliferation Kit, FLUOS works.
HB: Injecting BrdU into whole animalsfor in vivo labeling, then sacrificing theanimal and preparing tissue sections.Note: Only proliferating cells incorporate BrdU into their DNA.2Fixing BrdU-labeled tissue or cells.3Denaturing cellular DNA with acid.4Detecting incorporated BrdU withfluorescein-labeled anti-BrdU monoclonal antibody.5Analyzing the antibody-labeled samples with a flow cytometer or a fluorescence microscope.Specificity: The antibody conjugate (antiBrdU-fluorescein, F(ab’)2 fragments) willbind to BrdU-labeled DNA after the DNAis denatured and partially degraded withacid.
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