Apoptosis and Cell Proliferation, страница 10

Direct labeling withfluorescein-dUTP offers several other advantages. Direct labeling produces lessnonspecific background with sensitivityequal to indirect labeling (Figure 17) and,thus, is as powerful as the indirect methodin detecting apoptosis. Furthermore, thefluorescence may be converted into a colorimetric signal if an anti-fluorescein antibody conjugated with a reporter enzyme(Table 5) is added to the sample.Figure 17: Comparison of direct andindirect labeling of DNA strandbreaks in apoptotic cells. PBL wereincubated with 1 µM dexamethasonefor 24 h at 37°C and then labeled byTUNEL. Recordings were made at thesame photomultiplier settings.(Data were kindly provided by R. Sgonc,University of Innsbruck, Austria).Result: Direct labeling is as sensitive asindirect labeling, but produces lessnon-specific background.

EAlthough the enzymatic labeling methodsare time-consuming (due to multiple incubation and washing steps), they are verysensitive and specific21.Caution: One has to keep in mind thatthese methods are based on the detection ofDNA strand breaks. There are rare situations when apoptosis is induced withoutDNA degradation. Conversely, extensiveDNA degradation, even specific to the internucleosomal linker DNA, may accompany necrosis. Thus, one should always useanother independent assay, along with theTUNEL method, to confirm and characterize apoptosis.Roche Molecular Biochemicals 26offersthree kits for the detection of DNA strandbreaks that occur during cell death. Each isdescribed on the following pages.Note: For technical tips on the TUNELmethod, see page 105 of the Appendix.Direct labeling procedure usingFluorescein-dUTPIndirect labeling procedure usingDIG-dUTP and anti-DIG-fluoresceinBACell number1Cell numberCell Death – Apoptosis and NecrosisApoptosis Assay Methods10 010 110 210 310 4Fluorescence intensitynon-apoptotic cells10 010 110 210 310 4Fluorescence intensityapoptotic cellsMethod/BM productLabelIndirect (secondary)detection systemAnalysis byIn Situ Cell DeathDetection Kit,FluoresceinFluorescein-dUTPNone (direct detection)Flow cytometryFluorescence microscopyIn Situ Cell DeathDetection Kit, APFluorescein-dUTPAnti-Fluorescein-APLight microscopyIn Situ Cell DeathDetection Kit, PODFluorescein-dUTPAnti-Fluorescein-PODLight microscopyG Table 5: Three different kits for the enzymatic labeling of DNA and the secondary detection systems required.26In Situ Cell Death Detection Kit, FluoresceinCat.

No. 1 684 79550 testsTypeDirect TUNEL labeling assayUseful forDetection of DNA strand breaks in apoptotic cells by flow cytometry or fluorescence microscopySamplesCells in suspension, adherent cells, cell smears, frozen or paraffin-embeddedtissue sectionsMethodEnd-labeling of DNA with fluorescein-dUTP, followed by direct analysis offluorescent cellsTime1–2 h (+ sample preparation, permeabilization, etc.)Significance of kit: The In Situ Cell DeathDetection Kit, Fluorescein measures andquantitates cell death (apoptosis) by labeling and detection of DNA strand breaksin individual cells by flow cytometry orfluorescence microscopy.

The kit offersa direct TUNEL detection method, formaximum sensitivity and minimal background.1Fixing and permeabilizing apoptoticcells.2Incubating the cells with the TUNELreaction mixture containing TdT andfluorescein-dUTP. During this incubation step, TdT catalyzes the attachmentof fluorescein-dUTP to free 3’OH endsin the DNA.Test principle: The assay uses an optimizedterminal transferase (TdT) to label free3’OH ends in genomic DNA with fluorescein-dUTP. The procedure involves:3Visualizing the incorporated fluoresceinwith a flow cytometer and/or a fluorescence microscope.1For a detailed overview of the steps in theprocedure, see Flow Chart 5.Cell suspensionsAdherent cells, cellsmears, cytospinsFrozen tissue sectionFix samples (1 h, RT)Formalin-fixed, paraffinembedded tissue sectionsF Flow Chart 5: Assay procedure,In Situ Cell Death Detection Kit,Fluorescein.Dewax, rehydrate, andtreat with proteaseWash samplesPermeabilize samples (2 min, on ice)Wash samplesIncubate with TUNEL reaction mixture [enzyme solution + labeling solution] (60 min, 37°C)Wash samplesAnalyze by flowcytometry or fluorescencemicroscopyCell Death – Apoptosis and NecrosisApoptosis Assay MethodsThe TUNEL enzymatic labeling assayAnalyze by fluorescence microscopy27Cell Death – Apoptosis and NecrosisApoptosis Assay MethodsThe TUNEL enzymatic labeling assaySensitivity: The enzymatic labeling allowsthe detection of an apoptotic event that occurs, prior to changes in morphology andeven before DNA fragments become detectable in the cytoplasm22.Specificity: The amount of DNA strandbreaks in apoptotic cells is so large that thedegree of cell labeling in these assays is anadequate discriminator between apoptoticand necrotic cells19.1Can be used to assay:P Cells in suspension (permanent celllines, normal and tumor cells ex vivo)P Cytospins, cell smearsP Adherent cells cultured on chamberslidesP Frozen tissue sectionsG Figure 19: Detection of apoptotic cells (green) byfluorescence microscopy in a tissue section fromrat.

A tissue section from a rat spinal cord was preparedand assayed with the In Situ Cell Death Detection Kit,Fluorescein. The treated section was viewed under afluorescence microscope. (Photomicrograph was kindlyprovided by R. Gold, University of Würzburg, Germany.)Result: A subpopulation of apoptotic cells, scatteredthroughout the tissue section, are intensely stained(green) by the TUNEL treatment and are easily visibleunder the microscope.P Formalin-fixed, paraffin-embedded tissue sectionsKit contents1.

Enzyme solution (TdT), 5 tubes2. Labeling solution (nucleotide mix),5 tubesOther applications: For more examples ofhow the In Situ Cell Death Detection Kitcan be used in the lab, see Appendix, page122.Typical results: See Figures 18–19.Technical tips: For more information onthe use of the kit for flow cytometric analysis, see page 111 in the Appendix of thisguide.G Figure 18: Detection of apoptotic cells by flow cytometry using the In Situ Cell Death Detection Kit,Fluorescein. HL60 cells were cultured in the absence (A) or presence (B) of 2 µg/ml Camptothecin for 3 h at 37°C.Cells were incubated either with TUNEL reaction mixture (m) or label solution (M) or PBS for autofluorescence (m).28In Situ Cell Death Detection Kit, APCat.

No. 1 684 80950 testsIn Situ Cell Death Detection Kit, PODCat. No. 1 684 81750 testsTypeIndirect TUNEL labeling assayUseful forDetection of DNA strand breaks in apoptotic cells under a light microscopeSamplesCell smears, adherent cells, cytospins, frozen or fixed tissue sectionsMethodEnd-labeling of DNA with fluorescein-dUTP, followed by detection of incorporated fluorescein with an antibody and visualization of the antibodyTimeApprox.

3 h (+ sample preparation, permeabilization, etc.)Significance of kits: These two In Situ CellDeath Detection Kits measure cell death(apoptosis) by detecting DNA strandbreaks in individual cells by light microscopy. The AP and POD kits offer an indirectTUNEL detection method, which is a fast,sensitive, and specific light microscopicassay.Test principle: The assay uses an optimizedterminal transferase (TdT) to label free3’OH ends in genomic DNA with fluorescein-dUTP. The procedure involves:1231Remove tissue or organ and use standard methods to process for:Frozen sectioningParaffin embeddingFix sections in formalin (30 min, RT)Dewax, rehydrate sections by standard methodsOptional: Inactivate endogenous POD/AP activityWash slidesFix section (30 min, RT)Add protease and incubate (30 min, 37°C)Fixing and permeabilizing apoptoticcells or tissue sections.Incubating the cells with the TUNELreaction mixture containing TdT andfluorescein-dUTP.

During this incubation step, TdT catalyzes the attachmentof fluorescein-dUTP to free 3’OH endsin the DNA.Detecting the incorporated fluoresceinwith an anti-fluorescein antibody APconjugate (In Situ Cell Death DetectionKit, AP) or an anti-fluorescein antibodyPOD conjugate (In Situ Cell Death Detection Kit, POD).Wash slidesPermeabilize sections (2 min, on ice)Add TUNEL-reaction mixture and incubate (60 min, 37°C)Wash slidesOptional: Analyze by fluorescence microscopyAdd Anti-Fluorescein-AP or -POD and incubate (30 min, 37°C)Wash slides4Visualizing the immunocomplexed APor POD with a substrate reaction.Cell Death – Apoptosis and NecrosisApoptosis Assay MethodsThe TUNEL enzymatic labeling assayAdd substrate and incubate (5–20 min, RT)Analyze by lightmicroscopyG Flow Chart 6: Assay procedure In Situ Cell Death Detection Kits (AP or POD).29Cell Death – Apoptosis and Necrosis1Apoptosis Assay MethodsThe TUNEL enzymatic labeling assaySensitivity: The enzymatic labeling ofDNA strand breaks allows the detection ofan early apoptotic event.

This is especiallyimportant if apoptosis is studied in vivo,e.g. in tissue sections, since apoptotic cellsare rapidly and efficiently removed in vivo.Specificity: The amount of DNA strandbreaks in apoptotic cells is so large that thedegree of cell labeling in these assays is anadequate discriminator between apoptoticand necrotic cells.Figure 20: Detection of apoptoticcells by TUNEL and peroxidasestaining in rabbit endometrium. A tissue section from rabbit endometriumwas prepared and assayed with the InSitu Cell Death Detection Kit, POD. Slidewas counterstained with hematoxylinand viewed under a light microscope.Result: A subpopulation of apoptoticcells, scattered throughout the tissuesection, are intensely stained (brown)by the TUNEL treatment and subsequent peroxidase immunostaining.

ECan be used to assay:P Cells smears, adherent cellsP CytospinsP Tissue sections (frozen or paraffin-embedded).Kit contentsIn Situ Cell Death Detection Kit, AP1. Enzyme solution (TdT), 5 tubes2. Labeling solution (nucleotide mix),5 tubes3. Anti-Fluorescein-AP conjugate, readyto useIn Situ Cell Death Detection Kit, POD1. Enzyme solution (TdT), 5 tubes2.

Labeling solution (nucleotide mix),5 tubes3. Anti-Fluorescein-POD conjugate,ready to useFigure 21: Detection of apoptoticcells by TUNEL and alkaline phosphatase staining in rat spinal cord. Atissue section from rat spinal cord wasprepared and assayed with the In SituCell Death Detection Kit, AP. The slidewas viewed under a light microscope(Panel A). After viewing, the same slidewas stained with propidium iodide andviewed by fluorescence microscopy(Panel B).Result: A few apoptotic cells (red) areclearly visible after TUNEL treatmentand subsequent alkaline phosphataseimmunostaining (Panel A).

However, theapoptotic cells are not visible in thesame slide after staining with propidiumiodide (Panel B). EANote: For added flexibility and convenience, the components of these kits, as wellas several AP and POD precipitating substrates are also available as single reagents(Table 8).Typical results: See Figures 20–21.Technical tips: For more information onthe use of the kits for light microscopicanalysis, see page 107 in the Appendix, ofthis guide.BOther applications: For more examples ofhow the In Situ Cell Death Detection Kitscan be used in the lab, see Appendix, page122 .For your convenience, we offer a numberof additional single reagents to optimizeyour TUNEL reaction (Table 6).ProductCat.