Apoptosis and Cell Proliferation, страница 7

Take an aliquot ofthe supernatant (cell lysate) and determine the amount ofapoptotic nucleosomes present.normalcellnecroticcellIncubate supernatant with immunoreagent(containing anti-histone and anti-DNA) (2 h, RT)apoptoticcellWash MTP wells three times with incubationbuffer at RTBPODAdd substrate solution to wells and incubate(approx. 15 min, RT)PODMeasure absorbance at 405 nmStreptavidincoated MTPAnti-histoneBiotinSample ContainingNucleosomesAnti-DNAPODABTSTSubstrateFigure 7: How the Cell Death Detection ELISAPLUS works.GPanel A: Sample preparationPanel B: ELISASensitivity: In a model system, nucleosomes were detectable in as few as 600 campothecin-induced U937 cells (Figure 8).However, the lower limit for detecting dying/dead cells in a particular sample varieswith the kinetics of the apoptotic process,the cytotoxic agent used, and the numberof affected cells in the total cell population.G Flow Chart 2: Assay procedure,Cell Death Detection ELISAPLUS.321010100100010000cells / wellG Figure 8: Sensitivity of Cell Death DetectionELISAPLUS.

Different cell concentrations of U937 cellswere incubated with CAM (2 µg/ml) or without CAMfor 4 h at 37°C. 20 µl of cell culture supernatant andcell lysates were analyzed in the ELISA. Substratereaction time: 10 min. v Lysate with CAM, v Lysatewithout CAM, m Supernatant with CAM, m Supernatantwithout CAMResult: The ELISA can clearly detect apoptosis-relatednucleosomes in as few as 600 cells.14P Adherent cellsP Cells in suspension cultureP Cell culture supernatantP Lysates of cells obtained ex vivoP Serum, or plasmaKit contents1.

Anti-histone antibody (clone H11-4),biotin-labeled2. Anti-DNA antibody (clone M-CA-33),peroxidase-conjugated3. DNA-histone complex (positive control)4. Incubation buffer, ready-to-use5. Lysis buffer, ready-to-use6. Substrate buffer, ready-to-use7. ABTS® substrate tablets8. MTP modules (12 x 8 wells)9. Adhesive plate cover6241enrichmentCan be used to assay:83absorbance [A 405 nm–A 480 nm]Specificity: The ELISA is specific for nucleosomes containing single- or doublestranded DNA (Figure 9).

It is not speciesspecific.20100.010.1CAM [µg/ml]1Cell Death – Apoptosis and NecrosisApoptosis Assay MethodsAssays that measure DNA fragmentation10G Figure 9: Dose-response experiment analyzedby the Cell Death Detection ELISAPLUS. U937 cells(104 cells/well, in 200 µl) were incubated with differentconcentrations of CAM for 4 h at 37°C. Before and afterlysis, cells were centrifuged and a 20 µl aliquot of thesupernatant was analyzed with the Cell Death DetectionELISAPLUS.

Results were plotted as dose vs. response.Substrate reaction time: 5 min. l Lysate, m Supernatant, v Enrichment factor of the lysate.Result: Amounts of cytoplasmic oligonucleosomes (anindicator of apoptosis) increase as CAM concentrationincreases. Cell culture supernatants removed from thecells after treatment (but before lysis) gave no signal,indicating that there are no necrotic cells during thetreatment.Typical results: see Figure 9Other applications: For more examples ofhow the Cell Death Detection ELISAPLUScan be used in the lab, see Appendix, page121.15Cell Death – Apoptosis and NecrosisApoptosis Assay MethodsAssays that measure apoptosis-induced proteases (caspases)1.2.1.2 Assays that measure apoptosisinduced proteases (caspases)Several caspases (see Table 25, in the Appendix, page 115) are thought to mediatevery early stages of apoptosis10. For instance, one of these, caspase 3 (CPP32) isrequired for the induction of apoptosis bycertain effectors [especially tumor necrosisfactor and the cytotoxic T cell ligand effector, CD95 (also called Fas)] Enari et al.(1996) , Nature 380, 723–726.1These proteases cleave numerous substrates at the carboxy site of an aspartateresidue.

All are synthesized as pro-enzymes; activation involves cleavage at aspartate residues that could themselves besites for the caspase family. As caspasesare probably the most important effectormolecules for triggering the biochemicalevents which lead to apoptotic cell death,assays for determination of caspase activation can detect apoptosis earlier than manyother commonly used methods.16The most elucidatory assay for these caspases involves western blot detection of proteolytic cleavage products found in apoptotic cells. An antibody, Anti-PARP, sold byRoche Molecular Biochemicals, can beused in such an assay.

The antibody can detect intact and cleaved forms of Poly-ADPRibose Polymerase, a target for some caspases.For specific and quantitative measurementof caspase activity Western blotting is notsuitable. To quantify caspase activation enzyme activity assays based on detection ofcleaved caspase substrates have been developed recently. However most of the caspase substrates are not exclusively cleavedby a specific caspase but only preferentially, while other members of the caspasesfamily act on these substrates to a lowerextent.

Roche Molecular Biochemicals offers a casapase 3 activity assay with highestspecificity by the use of an immunosorbentenzyme assay principle.Caspase 3 Activity AssayCat. No. 2 012 95296 testsTypeImmunosorbent enzyme assay, fluorometricUseful forSpecific, quantitative in vitro determination of caspase 3 activitySamplesCell lysates, recombinant caspase 3 (CPP32)MethodCell lysis, followed by capturing of caspase 3 by a specific antibody and fluorometric determination of proteolytic cleavage of the substrateTimeApprox. 5 h (after induction of apoptosis)Significance of kit: This kit allows specific,quantitative detection of caspase 3 activityin cellular lysates after induction of apoptosis. Caspase 3 activation seems to play akey role in initiation of cellular events during the apoptotic process.

The immunosorbent enzyme assay principle of this kitguarantees high specificity without crossreactions with other known caspases. Thefluorochrome generated by proteolyticcleavage of the caspase substrate is proportional to the concentration of activated caspase 3 in the lysates.5Washing the immobilized antibodycaspase 3 complexes three times to remove cell components that are not immunoreactive.6Incubating sample with caspase substrate (Ac-DEVD-AFC) that is proteolytically cleaved into free fluorescentAFC.7Measuring generated AFC fluorometrically.1Test principle: The assay uses a fluorometric immunosorbent enzyme assay(FIENA) principle. The procedure (Figure 10 and Flow Chart 3) involves:12Inducing apoptosis in cells by desiredmethod (for instance 2 x 106 cells).

Afterthe induction, the cells are washed andpelleted by centrifugation.Preparing samples by resuspending andincubating cells in lysis buffer. After lysis and following centrifugation, samples can be removed for direct analysisor storage.3Coating microtiter plate with anti-caspase 3 solution and blocking of unspecific binding.4Transferring a sample to the anti-caspase 3-coated well of a microtiter plateand capturing of caspase 3.anti-caspase 3coated MTPsample containingactivated caspase 3Cell Death – Apoptosis and NecrosisApoptosis Assay MethodsAssays that measure apoptosis-induced proteases (caspases)Ac-DEVD-AFCfluorescencesubstrateG Figure 10: How the Caspase 3 Activity Assay works.17Cell Death – Apoptosis and NecrosisApoptosis Assay MethodsAssays that measure apoptosis-induced proteases (caspases)Sample preparationTreat sample (2 x 106 cells) with apoptosis-inducingagent. Include a negative control without induction.(1–24 h)Wash treated and control cells with ice-cold PBS andcentrifuge (300 x g ) (5 min, RT)Incubate cell pellet in lysis buffer (1 min, on ice)1Centrifuge at maximum speed in a tabletop centrifuge(1 min/RT)Remove 100 µl sample for direct analysis or storagefor 1 week at –20°CCoating microtiter plate (MTP)Incubate MTP with anti-caspase 3 coating solution(either at 37°C for 1h or at 4°C over night)Block unspecific binding by incubation with blockingbuffer (30 min, RT)Remove blocking solution and wash 3 times withincubation buffer (3 x 1 min, RT)Assaying protease activityAdd sample (100 µl lysate, positive control) intoMTP well (1h, 37°C)Remove sample and wash 3 times with incubationbuffer (3 x 1 min, RT)Add freshly prepared substrate solution (1–3 h, 37°C)Sensitivity: In a model system, caspase 3activity was clearly detectable in lysates of106 cells with 5 % apoptotic cells (Figure11).

However, the lower limit for determination of caspase 3 activity in cellular lysates of dying cells in a particular samplevaries with the kinetics of the apoptoticprocess, the apoptotic agent used, and thenumber of affected cells within the total cellpopulation.Specificity: This fluorometric immunosorbent enzyme assay is highly specific forcaspase 3 by the use of an anti-caspase 3specific monoclonal capture antibody incombination with a specific caspase substrate. Enzyme activity of natural and recombinant human caspase 3 is detected bythis assay.