Apoptosis and Cell Proliferation (522915), страница 12
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10 min, RT)Incubate washed cells with streptavidin/avidin conjugate (20 min, 4°C)1Centrifuge stained cells (200 x g ) and wash once with incubation buffer (approx. 10 min, RT)Add incubation buffer to stained cells Analyze by fluorescence microscopyAnalyze by flow cytometryCell Death – Apoptosis and NecrosisApoptosis Assay MethodsAssays that measure membrane alterationsAdd substrate (5–15 min, RT)and washAnalyze by light microscopySpecificity: Annexin-V-Biotin binds apoptotic cells and leaky necrotic cells.Can be used to assay:P Cell lines (adherent/suspensions)P Freshly isolated cellsReagent contents:P Annexin-V-Biotin solution, 50 x concentrated.Typical results: See Figure 25.FLUOSProductApplicationCat. No.Pack SizeAvidin-Fluoresceinfluorescence microscopy, flow cytometry1 975 5951 mgSA-R-Phycoerythrinfluorescence microscopy, flow cytometry1 428 56050 µg (1 ml)Avidin-Rhodaminefluorescence microscopy, flow cytometry1 975 6091 mgSA-AMCAfluorescence microscopy, flow cytometry1 428 5781 mgSA-Peroxidaselight microscopy1 089 153500 U (1 ml)SA-Alkaline Phosphataselight microscopy1 089 1611000 U (1 ml)SA-b-Galactosidaselight microscopy1 112 481500 UF Figure 25: Flow cytometric analysis of apoptotic U937 cells stainedwith Annexin-V-Biotin, StreptavidinFLUOS and propidium iodide.
U937cells (a leukemic cell line) were cultivated for 4 h with 4 µg/ml campothecin.Cells were stained with Annexin-VBiotin and propidium iodide, then incubated with Streptavidin-fluorescein(SA-FLUOS) and analyzed. Single parameter histograms are shown at thetop (Annexin-V-Biotin/SA-FLUOS) andon the right side (PI) of the diagram.Two parameter histograms are shownin quadrants 1–4.
PI, propidium iodide;FLUOS, fluorescein.Result: Flow cytometric analysis clearlydifferentiates normal cells (quadrant 3)with low FLUOS and low PI staining,apoptotic cells (quadrant 4) with highFLUOS and low PI staining, and necroticcells (quadrant 2) with high FLUOS andhigh PI staining.G Table 8: Streptavidin (SA) conjugates available for the indirect assay of apoptotic cells with Annexin-V-Biotin.Note: Additional substrates can be found in Table 6.35Cell Death – Apoptosis and NecrosisApoptosis Assay MethodsAssays that use DNA stains1.2.2.3 Assays that use DNA stainsOne can differentiate between three methods for studying cell death that use DNAstains: dye exclusion method, profile ofDNA content, morphological changes.Dye exclusion methodViable (intact plasma membrane) and dead(damaged plasma membrane) cells canbe discriminated by differential staining.Cells with disturbed plasma membranepermeability are stained, whereas undamaged (viable) cells are not stained with dyesthat do not penetrate the plasma membrane(“exclusion dyes”).
The most frequentlyused dye for exclusion tests is trypan blue.In addition, the fluorescent dye, propidiumiodide (PI) which becomes highly fluorescent after binding to DNA, can be usedin the same manner. The stained and unstained cells are counted with a standardlight microscope (trypan blue), or flow cytometer (PI) (Table 9).1DNA-binding dyes(Fluorochromes)Profile of DNA contentIf cells are permeabilized, the LMW DNAinside the cytoplasm of apoptotic cellsleaks out during the subsequent rinse andstaining procedure. The lower DNA content of these cells means they contain lessDNA stained by the fluorochrome.
Thus,cells with lower DNA staining than thatof G1 cells (the so-called “sub-G1 peaks”,“A0” cells) have been considered apoptotic.The reduction in staining/DNA content ofthese cells is measured by flow cytometry(Figure 26). The major disadvantage of thistechnique is that apoptotic G2-Phase cellsexhibit a reduced DNA content, whichcould represent the DNA content of a G1cell. Therefore it may not be detected asapoptotic. This would result in an underestimation of the apoptotic population.Dye entersDye stainsViable cellsNon viable cellsNucleus (DNA)Cytoplasm (RNA)Acridine orangeYesYesGreenRed-orangeHoechst 33342YesYesBlueNoHoechst 33258NoYesBlueNoDAPINoYesBright blueNoEthidium bromideNoYesOrangeSlightly redPropidium iodideNoYesRedNoG Table 9: Common fluorochromes used to stain the genomic DNA of viable and/or non-viable cells.Figure 26: Typical flow cytometricprofile of the DNA content in normal (A)and apoptotic cells (B), stained with PI.Result: A prominent “sub-G1” peak(earliest peak) appears in apoptoticcells, but not in normal cells.
E36G1S G2/MApoptotic G1AS G2/MBMorphological changesOn the other hand, the bisbenzimidazoledye, Hoechst 33342 (and also acridineorange), penetrates the plasma membraneand stains DNA in cells; without permeabilization. In contrast to normal cells, thenuclei of apoptotic cells have highly condensed chromatin that is uniformly stainedby Hoechst 33342. This can take the formof crescents around the periphery of thenucleus, or the entire nucleus can appearto be one or a group of featureless, brightspherical beads. These morphologicalchanges in the nuclei of apoptotic cells maybe visualized by fluorescence microscopy.They are also visible in permeabilized apoptotic cells stained with other DNA bindingdyes like DAPI (Figure 27).Dive et al.30 have reported that during ashort exposure to Hoechst 33342, apoptotic cells have stronger blue fluorescencecompared to non-apoptotic cells.
Co-staining of the cells with PI allows the discrimination of dead cells from apoptotic cells. If7-amino-actinomycin is used instead of PI,cell surface antigens immunostained withfluorescein and phycoerythrin may bequantitated simultaneously31.One drawback of using any vital stainingmethod for measuring apoptosis is the variability of active dye uptake in differentcells and its possible change during certain treatments.
Therefore, the ability ofHoechst 33342 to discriminate apoptoticcells from normal cells by increased uptakeof dye has to be tested for each new cellsystem31.ReagentCat. No.Pack sizeFluorescenceTypical resultsPropidium iodide*1 348 63920 mlred orangeSee Table 9 and Figure 24DAPI4’,6-Diamidine-2’-phenylindoledihydrochloride236 27610 mgblueSee Table 9 and Figure 27Ethidium bromide200 2712gorangeSee Table 9 and Figure 281G Table 10: Fluorescent dyes that stain double-stranded DNA*Only sold in the USG Figure 27: Fluorescent microscopic analysis ofapoptotic cells stained with DAPI. DAPI stains thenuclei of all cells (blue).Result: The characteristic condensed nuclei of apoptoticcells are clearly visible here.Cell Death – Apoptosis and NecrosisApoptosis Assay MethodsAssays that use DNA stainsG Figure 28: Fluorescent microscopic analysis ofmitotic cells stained with ethidium bromide.
DNAwas stained with ethidium bromide (orange). Mitoticspindles were stained with anti-tubulin antibody (green).Result: Mitotic cells (with condensed DNA) are brightlystained. Without the double stain, mitotic cells could bemistaken for apoptotic cells, since both have condensedDNA.37Summary of methods for studying apoptosis in individual cellsSummary of methods for studying apoptosis in individual cells1.2.2.4 Summary of methods for studying apoptosis in individual cells.Method/Roche Molecular Biochemicals productParameter analyzedAssay principleAdvantagesLimitationsStaining of chromosomal DNA afterpermeabilization (DNA content)22, 32DNA fragmentation(HMW DNA, DNA content)P Apoptotic cells are permeabilized with ethanol or detergent.
Duringthis procedure the LMW DNA inside the apoptotic cell leaks out andis removed during the subsequent washing steps.P The HWM DNA retained in the cells is stained with a DNA binding dyesuch as propidium iodide.P The amount of HMW DNA is quantified by flow cytometry(“sub G1” or “A0” peak).P Quick and cheapP Minimal overlap between the peak representing apoptotic (“sub G1”)and normal G1 cellsP Estimation of position in cell cycle allows cell cycle specificity ofapoptosis to be studiedP Applicability to any DNA fluorochrome and instrumentP Degree of extraction of LMW DNA during the washing and stainingpage 36procedure not always reproducibleof this guideP Not specific for apoptosis: Sub G1 peak can also represent mechanically damaged cells, cells with different chromatin structure, or normal cells with lower DNA content in heterogeneous cell populationsP May not detect cells induced to apoptosis in G2P No discrimination of apoptotic cells from dead cells which have losttheir membrane integrityStaining of chromosomal DNAChromatin morphologyP Apoptotic cells are stained by the addition of DNA fluorochromeswhich are able to cross the intact plasma membrane, such asacridine orange.
Can be done with DAPI on fixed cells.P The stained DNA allows the altered morphology of the nuclearchromatin to be visualized by fluorescence microscopy.P Quick and cheapP Discrimination between viable and dead cells when counterstainedwith propidium iodide using vital dyes (acridine orange,Hoechst 33342)P Simultaneous staining of cell surface antigens with standardfluorescein and phycoerythrin conjugates possible if Hoechst 33342is combined with 7-amino-actinomycin DP No quantitative measurementP Subjective: no clear cut-off point between normal and apoptotic cellsP Clear morphologically distinct apoptotic nuclei appear late duringapoptosis: May lead to an underestimation of apoptotic cellsActive labeling of cells by nick translation(ISNT)34DNA strand breaks (nicks) and DNAfragmentation (staggered DNA ends)P Apoptotic cells are fixed with formaldehyde and subsequentlypermeabilized.P DNA strand breaks are labeled with modified nucleotides usingexogenous DNA polymerase (nick translation).P The incorporated nucleotides are visualized with a secondarydetection system which has a reporter molecule (e.g.
fluorescein,AP, POD).P Counterstaining with DNA fluorochrome (profile of DNA content)allows cell cycle specificity of apoptosis to be studied (only by flowcytometry)P Identification of apoptosis at a molecular level (DNA strand breaks)P Suitable for tissue sectionsP Labor-intensive and time-consuming; only a few tests may beperformed simultaneouslyP Undefined cell loss during fixation procedure (loss of specific cellpopulation?)P Many cells (2–5 x 106/test) requiredActive labeling of cells by end labeling(TUNEL)20, 35DNA strand breaks (nicks) and DNAfragmentation (staggered DNA ends)P Apoptotic cells are fixed with formaldehyde and subsequentlypermeabilized.P DNA strand breaks are labeled with modified nucleotides usingexogenous terminal transferase (end labeling).P The incorporated nucleotides are visualized directly (e.g.
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