Apoptosis and Cell Proliferation (522915), страница 25
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As a measure ofFixed, BrdU-labeled sample (from Flow Chart 20 or Flow Chart 21)Cell Proliferation and ViabilityDenature sample DNA e.g., with HCl (10–20 min, RT) or microwaves (15 min, 100°C)Incubate sample withAnti-BrdU-FluoresceinIncubate sample withAnti-BrdUIncubate sample withAnti-BrdU-APIncubate sample withAnti-BrdU-PODAnalyze sample by flowcytometry or fluorescencemicroscopy.Incubate sample withanti-mouse-fluorescein oranti-mouse-enzyme(+ enzyme substrate)Add alkaline phosphatasesubstrate and incubateuntil color formsAdd peroxidase substrateand incubate until colorformsAnalyze sample by flowcytometry, fluorescence orlight microscopyAnalyze sample by light microscopyG Flow Chart 23: Immunostaining procedure, Anti-BrdU antibody and conjugates.Specificity: Conjugated or unconjugatedanti-BrdU antibody will bind to BrdU-labeled DNA after the DNA is denaturedand partially degraded (e.g., with DNase,acid or microwaves). The antibody specifically recognizes 5-bromo-2’-deoxyuridine;it shows no cross-reactivity with any endogenous cellular components such as thymidine or uridine.2Can be used to assay:P Cell lines (in adherent or suspension cellculture)P Freshly isolated cells, or tissue explantslabeled with BrdU in vitroP Frozen or paraffin-embedded tissuesections from animals labeled withBrdU in vivo.94apoptotic cells, fragmented DNA contentwas quantitated with the In Situ Cell DeathDetection Kit, Fluorescein (TUNEL method, according to the kit package insert).Either of two flow cytometric techniqueswas used to determine the cell cycle position of the cells: 1) Relative DNA contentwas determined by treating the cells with5 µg/ml propidium iodide and 200 µg/mlribonuclease (30 min, room temperature).2) Cells going through S-phase were identified by labeling with BrdU (10 µM BrdU,30 min), detection of BrdU-labeled cellswith anti-BrdU monoclonal antibody(30 min, 37°C), and visualization of thosecells with R-phycoerythrin-conjugatedgoat anti-mouse antibody (30 min, 37°C).For results, see Figure 67.Other applications: For examples of howthe Anti-BrdU conjugates and the antibodies may be used in the lab, see Appendix, page 126.Methods for studying cell proliferation and viability in individual cellsAssays that measure DNA synthesisRelative Angle Light ScatterCell Proliferation and ViabilityRelative Fluorescein-dUTP-contentFigure 67: Concomitant flow cytometric analysis ofapoptosis and cell cycle position with the anti-BrdUantibody, propidium iodide, and the In Situ CellDeath Detection Kit, Fluorescein.
Cultured mousethymocytes were treated with ionomycin (2 h or 12 h) toinduce apoptosis. After treatment, the cells were harvested, fixed, and analyzed for apoptosis and cell cycleposition by flow cytometry. Histograms A, C, and E showdata obtained from cells after 2 h treatment with ionomycin. Histograms B, D, and F show data obtained fromcells after 12 h treatment with ionomycin. Histograms Aand B show fluorescein intensity (green fluorescence)alone, a measure of DNA fragmentation. Histograms Cand D show a two-parameter analysis of fluoresceinintensity (green fluorescence, DNA fragmentation) andpropidium iodide intensity (red fluorescence, DNA content).
Histograms E and F show a two-parameter analysis of fluorescein intensity (green fluorescence, DNAfragmentation) and phycoerythrin intensity (orange fluorescence, BrdU content). The percentage of positive cellsis indicated in each panel. [Data from Hanon, E., Vanderplasschen, A. and Pastoret, P.-P. (1996) BiochemicaNo. 2, 25–27.]Result: The ionomycin-treated cells contained about13% apoptotic cells (histogram A) after 2 h and about29% apoptotic cells (histogram B) after 12 h exposure.Concomitant analysis of apoptosis and total DNA content(histograms C and D) showed that apoptotic cells contained about as much DNA as cells in G0/G1 or earlyS-phase. Concomitant analysis of apoptosis and BrdUcontent after 12 h ionomycin treatment (histogram F)showed that 6% of the apoptotic cells went throughS phase (that is, were positive for BrdU) while 21% ofapoptotic cells remained in G0/G1 (that is, were negativefor BrdU).
ERelative DNA content2Relative BrdU contentReagentRecommendedconcentration forDNA labelingCat. No.Pack size5-Bromo-2’-deoxyuridinetablets (50 mg BrdU/tablet)In vitro : 10 µM BrdU*In vivo : 10 mM BrdU**586 06450 tabletsG Table 19: Bromodeoxyuridine for DNA labeling* For in vitro labeling of cells or tissue, incubate samples with 10 µM BrdU, 0.5–1.5 h, 37°C.** For in vivo labeling, inject the animal intraperitoneally with a sterile solution containing 10 mM BrdU (1 ml/100 gbody weight), then sacrifice the animal 1–4 h later and prepare frozen or paraffin-embedded tissue sections.95Methods for studying cell proliferation and viability in individual cellsAssays that monitor expression of cell cycle-associated antigens2.2.2.2 Assays that monitor expressionof cell cycle-associated antigensMonoclonal antibodies directed against cellcycle antigens can identify cells which areactively cycling77.
In some cases, these antibodies can distinguish specific phases ofthe cell cycle. Antibodies against such antigens as Ki-67 are especially useful for theclinical assessment of cell proliferationby immunohistochemical techniques78–81.Furthermore, immunocytochemical analysis of cells by flow cytometry allows quantitation of cell proliferation69.Important monoclonal antibodies used tostudy cell proliferation and the cell cycleare summarized in Table 19 and 20.S-phaseMitoK i- 6 7PCNAMitotiInde cxG1RG2K i- 6 7Cell Proliferation and ViabilityBrdULabelingKi-67seG0G Figure 68: Phases of cell cycle.296Methods for studying cell proliferation and viability in individual cellsAssays that monitor expression of cell cycle-associated antigensMonoclonal antibodies to cell cycle-associatedantigensTypeMonoclonal antibodies, from mouseUseful forDetection of cell cycle-associated antigens which are expressed only in proliferating cellsSamplesParaformaldehyde-fixed cells in suspension (flow cytometry), cell smears,tissue sectionsMethodIncubation of samples with monoclonal antibody, followed by (if necessary)visualization of monoclonal antibody with secondary antibody.
Also uselfulfor immunohistochemistry and cytochemistry , for western blotting in populations of cells.TimeVariable (depending on sample and antibody used)Significance of antibodies: Several nuclearantigens [e.g. proliferating cell nuclear antigen (PCNA), Ki-67 and topoisomerase IIalpha (Ki-S1)] are expressed only in proliferating cells. They are absent in restingcells. Thus, antigens that recognize theseantigens may be used to selectively markproliferating cells in cell populations andtissue.
Depending on the sample and theantibody used, analysis can be by flow cytometry, fluorescence microscopy, or lightmicroscopy.Test principle: The antibodies listed in Table 20 may be used to detect nuclear antigens present only in proliferating cells. Theprocedure involves (Flow Chart 24):1Fixing cells or tissue so the target antigen is preserved. (See “Can be used toassay” next page for appropriate samples for each antibody.)2Detecting the target antigen with amonoclonal antibody.3(Optional) Localizing unconjugatedmonoclonal antibody with a secondaryanti-mouse antibody detection system.4Analyzing the antibody-labeled samples with a flow cytometer, a fluorescence microscope, or a light microscope.Fix cells with paraformaldehydePrepare frozen tissue sectionsPrepare formalin-fixed, paraffinembedded tissueIncubate with Anti-PCNA orAnti-Ki-67 (Ki-S5) or Anti-Topoisomerase II (30 min, RT)Incubate with Anti-PCNA orAnti-Ki-67 (Ki-S5) or Anti-Topoisomerase II (1 h, RT)Digest tissue section with trypsin(30 min, 37°C)Incubate with Anti-mouseIg-fluorescein* (30 min, RT)Incubate with bridging antibody(1 h, RT)Incubate with Anti-Topoisomerase II(1 h, RT)Analyze by flow cytometry orfluorescence microscopyIncubate with APAAP (1 h, RT)Incubate with bridging antibody(1 h, RT)Analyze by light microscopyIncubate with APAAP (1 h, RT)Cell Proliferation and Viability(See Table 20)2F Flow Chart 24: Immunostainingprocedure, unconjugated monoclonalantibodies to cell cycle-associatedantigens.Analyze by light microscopy* Anti-PCNA is also available as a fluorescein conjugate; If you are using this fluorescently labeled antibody,the incubation with fluorescently labeled anti-mouse antibody is not necessary.97Methods for studying cell proliferation and viability in individual cellsAssays that monitor expression of cell cycle-associated antigensSpecificity: All the monoclonal antibodiesto cell-cycle associated antigens are mousemonoclonal antibodies which react withnuclear antigens expressed only in proliferating cells.
They do not react with cytoplasmic antigens or with resting cells.Supplied as:Can be used to assay:Anti-Ki-67 (clone Ki-S5): 100 µg lyophilizateP Anti-Casein Kinase 2a (clone 1AD9):Frozen tissue sections and cell smears.P Anti-PCNA/Cyclin (clone 19F4): Paraformaldehyde-fixed cells and frozen tissue sections.P Anti-PCNA/Cyclin (clone PC10): Formalin-fixed and paraffin-embedded tissue sections.Anti-Casein Kinase 2a: 100 µg lyophilizateAnti-PCNA/Cyclin: 200 µg lyophilizateAnti-PCNA/Cyclin-Fluorescein: 200 µg in500 µl solutionAnti-Topoisomerase II-a (clone Ki-S1):100 µg lyophilizateAnti-Tranferrin Receptor, human: 200 µglyophilizate in 500 µl solution.Typical results: See Figures 69–70.Cell Proliferation and ViabilityP Anti-Ki-67 (clone Ki-S5): Paraformaldehyde-fixed cells, cell smears, formalinfixed or frozen tissue sections.P Anti-Topoisomerase II-a (clone Ki-S1):Paraformaldehyde-fixed cells in suspension, cell smears, formalin-fixed andparaffin-embedded or frozen tissue sections.P Anti-Tranferrin-Receptor, human (cloneB3/25): Paraformaldehyde-fixed cellsand paraffin-embedded tissue sections.298ProductSpecificityAnti-Casein Kinase 2a(clone 1AD9)The antibody reacts with casein kinase 2a, a growth related serine/threonine proteinkinase.
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