Apoptosis and Cell Proliferation (522915), страница 24
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The antibody specifically recognizes5-bromo-2’-deoxyuridine; it shows nocross-reactivity with any endogenous cellular components such as thymidine or uridine.Can be used to assay:P Cell lines (in adherent or suspension cellculture)P Freshly isolated cellsP Tissue explants labeled with BrdU invitroCells with partiallydenatured DNA,labeled with BrdU88Anti-BrdUFLUOSF(ab’)2-fragmentP Frozen or paraffin-embedded tissuesections from animals labeled withBrdU in vivo.Methods for studying cell proliferation and viability in individual cellsAssays that measure DNA synthesisKit contents1.
BrdU labeling reagent (1000 x), sterile2. Anti-BrdU-fluorescein, monoclonal,F(ab’)2 fragments3. Antibody incubation bufferTypical results: See Figures 61–63.G Figure 62: In vivo labeling and analysis of dorsal,Cell Proliferation and ViabilityG Figure 61: Flow cytometric measurement of totalDNA and incorporated BrdU with the In Situ CellProliferation Kit, FLUOS.
Exponentially growing U937cells were incubated with BrdU for 30 min. IncorporatedBrdU was measured flow cytometrically with the fluorescein-conjugated anti-BrdU antibody (<BrdU>fluos)from the In Situ Cell Proliferation Kit, FLUOS.
Total DNAwas counterstained with 1 µg/ml propidium iodide (PI).The phase of the cell cycle represented by each population of cells is indicated on the flow cytometric histogram. FL1-H, fluorescein intensity (relative BrdUcontent); FL3-H, propidium iodide intensity (relative DNAcontent).Result: BrdU labeling is confined exclusively to theS-phase (DNA synthesis) of the cell cycle.hyperproliferative epidermis tissue from mouse withthe In Situ Cell Proliferation Kit, FLUOS. UndilutedBrdU labeling solution from the kit was injected intraperitoneally into a mouse (1 ml BrdU solution/100 g bodyweight).
After 2 h of in vivo BrdU labeling, the mouse wassacrificed and 5 µm thick, paraffin-embedded tissuesections were prepared. Sections were deparaffinizedand rehydrated according to standard methods, thendigested with trypsin (15 min). DNA was partially denatured with HCl (20 min) and detected with anti-BrdUfluorescein. Each section was analyzed by differentialinterference microscopy (upper photo) and epifluorescence microscopy (lower photo). Magnification, 530 x.(Data kindly provided by S.
Kaiser and M. Blessing,I. Med. Klinik der Universität Mainz, Germany.)Result: Proliferating cells (green spots) are clearlyvisible throughout the tissue under epifluorescencemicroscopy.F Figure 63: In vitro labeling andanalysis of proliferating HeLa cellswith the In Situ Cell Proliferation Kit,FLUOS. HeLa cells in culture werelabeled with BrdU and the BrdU-labeledDNA detected with anti-BrdU-fluorescein, according to the package insert ofthe In Situ Cell Proliferation Kit, FLUOS.The labeled cell preparation was analyzed under a light microscope (upperphoto) and a fluorescence microscope(lower photo).Result: Proliferating cells (bright greennuclei) within the HeLa preparation areclearly visible under the fluorescencemicroscope.892Methods for studying cell proliferation and viability in individual cellsAssays that measure DNA synthesisIn Situ Cell Proliferation Kit, APCell Proliferation and ViabilityCat.
No. 1 758 7562100 testsTypeDirect immunostaining for light microscopyUseful 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. 2.5 h (+ 0.5–2 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 dividing or proliferating cells in vivo.
The InSitu Cell Proliferation Kit, AP can detectproliferating cells or tissues which havebeen tagged by in vitro or in vivo BrdU labeling. Labeled cells are analyzed by lightmicroscopy.Figure 64: How the In SituCell Proliferation Kit, AP works. HTest principle: The BrdU solution and alkaline phosphatase-conjugated anti-BrdUantibody supplied in the kit allow labeling and detection of proliferating cells.The procedure (Figure 64 and Flow Charts20–22) involves:1A: Incubating growing animal tissue orcells in vitro with BrdUorB: 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 orin a microwave oven.4Detecting incorporated BrdU with alkaline phosphatase-labeled anti-BrdUmonoclonal antibody.5Incubating the antibody-labeled preparation with an alkaline phosphatase substrate (Fast Red).6Analyzing the antibody-labeled samples with a light microscope.Specificity: The antibody conjugate [antiBrdU-fluorescein, F(ab’)2 fragments] willbind to BrdU-labeled DNA after the DNAis denatured.
The antibody specificallyrecognizes 5-bromo-2’-deoxyuridine; itshows no cross-reactivity with any endogenous cellular components such as thymidine or uridine.Fixed cells withpartially denaturedBrdU-labeled DNA90Anti-BrdU-APF(ab’)2-fragmentFast RedsubstrateMethods for studying cell proliferation and viability in individual cellsAssays that measure DNA synthesisCan be used to assay:P Cell lines (in adherent or suspension cellculture)P Freshly isolated cellsP Tissue explants labeled with BrdU invitroKit contents1.
BrdU labeling reagent (1000 x), sterile2. Anti-BrdU-alkaline phosphatase,monoclonal, F(ab’)2 fragments3. Antibody incubation buffer4. Fast Red substrate tablets5. Substrate bufferG Figure 66: Immunohistochemical detection ofproliferating cells in mouse colon with the In SituCell Proliferation Kit, AP. Paraffin-embedded tissuesections were prepared from BrdU-labeled mouse colonand the BrdU visualized with anti-BrdU-AP and Fast Redsubstrate, according to the package insert of the In SituCell Proliferation Kit, AP. Sections were analyzed under aphase-contrast light microscope.Result: Proliferating, BrdU-labeled cells (red stained nuclei) are visible in the mucosal crypts of the mouse colon.Cell Proliferation and ViabilityP Frozen or paraffin-embedded tissuesections from animals labeled withBrdU in vivo.Typical results: See Figures 65–66.Inject the animal with BrdU labeling reagentintraperitoneallySacrifice the animal (approx.
1–4 h later) and removetissue samples or organProcess tissue samples or organ for:G Figure 65: Immunohistochemical detection ofproliferating spermatogonia (undifferentiated stemcells) in mouse testes with the In Situ Cell Proliferation Kit, AP. Paraffin-embedded tissue sections wereprepared from BrdU-labeled mouse testis and the BrdUvisualized with anti-BrdU-AP and Fast Red substrate,according to the package insert of the In Situ CellProliferation Kit, AP. Sections were counterstained withhematoxylin and analyzed under a light microscope.Magnification, 630 x.Result: Proliferating, BrdU-labeled spermatogonia(red stained nuclei) are visible in the peripheral zone ofthe tissue.Frozen sectioningParaffin embeddingFreeze tissue samples/organ immediately afterremovalFix tissue samples/organin formalin immediatelyafter removal2Store sample frozen until Use standard dehydrationrequiredand paraffin embeddingfor sectioningprocedures to processfixed sampleCut sections of frozensample in a cryostatCut sections of embeddedsample on a microtomeTransfer sections onto aglass slide and fixTransfer sections onto aglass slide and use standard procedures to dewaxand rehydrate sectionsProceed with the immunostaining procedure(see Flow Chart 22)G Flow Chart 20: Assay procedure, in vivo labeling ofproliferating cells with BrdU.91Methods for studying cell proliferation and viability in individual cellsAssays that measure DNA synthesisAdherent cellsSuspension cellsTissue slicesGrow cells on cover slips/chamberslidesAdjust cell concentrationCut freshly isolated tissue sampleinto small piecesLabel cells/tissue slices with BrdU (0.5–1.5 h; 37°C)Remove BrdU labeling solution and wash cells/tissue slicesResuspend cells and preparecytospins or cell smearsProcess pieces for frozen sectioningor paraffin embeddingAir dry cells and fixProceed with fixing and dewaxing(see Flow Chart 20)Cell Proliferation and ViabilityAir dry cells and fixProceed with the immunostaining procedure (see Flow Chart 22)G Flow Chart 21: Assay procedure, in vitro labeling of proliferating cells with BrdU.Fixed, BrdU-labeled sample (from Flow Chart 20 or Flow Chart 21)2FLUOS KitAP KitDenature sample DNA with HCl(10–20 min, RT)Denature DNA with HCl (10–20 min, RT)or microwaves (15 min, 100°C)Incubate sample with anti-BrdU-fluorescein(30–45 min, 37°C)Incubate sample with anti-BrdU-AP(30 min, 37°C)Analyze cells by flowcytometryAnalyze cells/tissue byfluorescence microscopyAdd AP substrate and incubate until color forms(15–30 min, RT)Analyze by light microscopyG Flow Chart 22: Immunostaining procedure, In Situ Cell Proliferation Kits (FLUOS or AP).92Methods for studying cell proliferation and viability in individual cellsAssays that measure DNA synthesisAnti-BrdU, formalin gradeCat.
No. 1 170 37650 µgAnti-BrdU-FluoresceinCat. No. 1 202 69350 µgAnti-BrdU-AP, F(ab’)2 fragmentsCat. No. 1 758 74815 unitsCat. No. 1 585 860Cell Proliferation and ViabilityAnti-BrdU-Peroxidase, Fab fragment15 unitsTypeMonoclonal antibodies, from mouseUseful forDetection of BrdU-labeled DNA in proliferating individual cellsSamplesCultured or freshly isolated cells, tissue explants or sectionsMethodIncubation of samples with BrdU, followed by denaturation of DNA, detection of BrdU label with anti-BrdU antibody, and (if necessary) visualizationof anti-BrdU antibody with secondary antibodyTimeVariable (depending on sample and antibody used)A: Incubating growing animal tissue orcells in vitro with BrdU (available as aseparate reagent; see Table 19)orB: Injecting BrdU (available as a separate reagent; see Table 19) into wholeanimals for in vivo labeling, then sacrificing the animal and preparing tissuesections.Significance of antibodies: Bromodeoxyuridine (BrdU) is only incorporated into theDNA of proliferating cells.
Short periods(15–60 min) of incubation in vitro withBrdU will tag only cells going through theS phase of the cell cycle. Alternatively,BrdU can be injected into an animal to labelgrowing cells in vivo. Conjugated or unconjugated anti-BrdU antibody may beused to detect proliferating cells or tissueswhich have been tagged by in vitro orin vivo BrdU labeling. Depending on thesample and the antibody used, analysis canbe by flow cytometry, fluorescence microscopy, or light microscopy.12Fixing BrdU-labeled tissue or cells.Test principle: The anti-BrdU antibodiesmay be used to detect BrdU-labeled DNAin proliferating cells.
The procedure involves (Flow Chart 23):3Denaturing cellular DNA.4Detecting incorporated BrdU with conjugated or unconjugated anti-BrdUmonoclonal antibody.2Note: Only proliferating cells (cells inS-phase) incorporate BrdU into theirDNA.93Methods for studying cell proliferation and viability in individual cellsAssays that measure DNA synthesis56(Option) A: Localizing unconjugatedanti-BrdU antibody with a secondaryantibody detection systemor(Option) B: Localizing enzyme-conjugated anti-BrdU antibody with an enzyme substrate.Analyzing the antibody-labeled samples with a flow cytometer, a fluorescence microscope, or a light microscope.Typical results: The anti-BrdU antibodyhas been used to determine the cell cycleposition of apoptotic cells76.Briefly, the experimental procedure was asfollows: Cultured mouse thymocytes weretreated with 0.5 µM ionomycin (2 h or 12 h)to induce apoptosis. After treatment, thecells were harvested, fixed in paraformaldehyde and ethanol (two-step fixation), andanalyzed for apoptosis and cell cycle position by flow cytometry.
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