Apoptosis and Cell Proliferation (522915), страница 15
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2.5 hSignificance of kit: This kit uses antibodiesto quantitate the levels of human, mouse, orrat p53 in smear/plasma, in tumor tissue,or in tumor cell lines. Wild type p53 is aprotein that suppresses malignant transformation by inducing apoptosis. Mutationsof the p53 gene which increases its stabilityallow transformation (immortalization) ofthe cell and are quite commonly found inmalignancies. The p53 protein seems also tobe involved in cell death induced by cytotoxic drugs.Test principle: The assay uses a one-stepsandwich immunoassay (Figure 31) to detect wild-type and mutant p53.
The procedure (Flow Chart 13) involves:1Preparing sample (e.g., detergent lysisof cells, homogenization of tissue), followed by centrifugation.Streptavidincoated MTPBiotinlabeled MABG Figure 31: How the p53 pan ELISA works..48Sample/standard2Transferring an aliquot of the samplesupernatant to streptavidin-coated wellof a microtiter plate.3Binding p53 in the supernatant with twoantibodies, a biotin-labeled monoclonalanti-p53 (capture antibody), which ispre-bound to the streptavidin-coatedplate, and a peroxidase-labeled polyclonal anti-p53 (detection antibody).4Washing the immobilized antibody-p53complexes five times to remove samplecomponents that are not immunoreactive.5Incubating sample with peroxidase substrate (tetramethylbenzidine, TMB).6Determining the amount of coloredproduct (and thus, of immobilized antibody-p53 complexes) spectrophotometrically.PODconjugated PABTMBsubstratePrepare sample (for example, cell lysate or tissuehomogenate)Centrifuge sample (10,000 x g ) (10 min, RT)Transfer aliquot of supernatant to Anti-p53 precoatedMTPSpecificity: The biotin-labeled capture antibody from mouse recognizes a conserved,pantropic, denaturation stable antigenicdeterminant of the p53 protein (human,mouse, rat).
The peroxidase-labeled detection antibody is highly specific for wildtype and mutant p53 from different species.Can be used to assay:Incubate supernatant with Anti-p53-peroxidase(1:15 diluted) (2 h, RT)Wash MTP wells five times with washing buffer(5 x 1 min, RT)Add substrate solution to wells and incubate(approx. 10–20 min, RT)Measure absorbance at370 nmAdd stop reagent to wells(1 min, RT)Measure absorbance at450 nmG Flow Chart 13: Assay procedure, p53 pan ELISA.Sensitivity: In 4 independent assays, thelower limit of detection for the assay wasdetermined to be 9 pg/ml.P Tissue homogenatesP Cell lysatesCell Death – Apoptosis and NecrosisApoptosis Assay MethodsDetection of apoptosis-related proteins1P Serum or plasmaKit contents1.
Anti-human-p53 pan, polyclonal, peroxidase-labeled2. Human p53 standards (six)3. Incubation buffer/Sample diluent,ready-to-use4. Washing buffer, 10 x concentrated5. TMB substrate solution6. TMB stop solution7. Streptavidin-coated microtiter plate,precoated with monoclonal Anti-p53(biotin-labeled)8. Adhesive plate cover foilsNote: The peroxidase-labeled antibody inthis kit, as well as other antibodies to p53are available as separate reagents see page46.49Cell Death – Apoptosis and NecrosisCytotoxicity Assay MethodsRelationship between cytotoxicity, apoptosis and necrosis1.3 Cytotoxicity Assay Methods1.3.1 Relationship between cytotoxicity, apoptosis and necrosisAs discussed in Section 1.1.1 of this guide,there are two experimentally distinguishable mechanisms of cell death: necrosis, the“accidental” cell death that occurs whencells are exposed to a serious physical orchemical insult, and apoptosis, the “normal” cell death that removes unwanted oruseless cells.1In contrast to these two cell death processes, cytotoxicity does not define a specificcellular death mechanism.
Rather, cytotoxicity is simply the cell-killing propertyof a chemical compound (such as a food,cosmetic, or pharmaceutical) or a mediatorcell (such as a cytotoxic T cell), independent from the mechanisms of death.Note: Cytotoxicity may also be used, as it isin this guide, to denote a laboratory methodfor detecting dead cells, regardless of themechanism of their death.Figure 32: Schematic illustration of thethree basic principles to assess plasmamembrane leakage.HExample of cytotoxicityA common example of cytotoxicity is cellmediated cytotoxicity.
Cells of the immunesystem [such as cytotoxic T cells, natural killer (NK) cells, and lymphokine-activated(LAK) cells] can recognize and destroy damaged, infected and mutated target cells.Although the recognition machinery usedby these cells is very different, their mechanism of target cell destruction may be verysimilar.Uptake of dyes (e.g. Trypan blue, propidium iodide)[ 51 Cr ][3H]Release of artificial label (e.g. [ 51 Cr], [ 3 H])Release of cytoplasmic enzymes (e.g. LDH)50Two possible cytocidal mechanisms havebeen proposed for cell-mediated cytotoxicity: (i) the apoptotic mechanism, in whichthe effector cell triggers an autolytic cascade in the target cell and the genomicDNA fragments before cell lysis; and (ii)the lytic mechanism, in which lytic molecules, notably perforin, are secreted by theeffector cell into the intercellular space andpolymerize to form pores in the target cellmembrane, leading to cell lysis3, 47. Thesetwo mechanisms are not mutually exclusiveand, quite possibly, are complementary.1.3.2 Methods for studyingcytotoxicityMost current assays for measuring cytotoxicity are based on alterations of plasmamembrane permeability and the consequent release (leakage) of components intothe supernatant or the uptake of dyes, normally excluded by viable cells (Figure 32)(see also 1.2.2.3, on page 36 “dye exclusionmethod”).
A serious disadvantage of suchpermeability assays is that the initial sites ofdamage of many, if not most cytotoxicagents are intracellular. Therefore, cellsmay be irreversibly damaged and committed to die and the plasma membrane is stillintact. Thus, these assays tend to underestimate cellular damage when compared toother methods.
Despite this fact, some permeability assays have been widely acceptedfor the measurement of cytotoxicity.Alternatively, dead cells are unable to metabolize various tetrazolium salts (see alsoSection 2.2.1.1). This allows the use of thecolorimetric assays MTT, XTT, or WST-1to measure cell survival. Apoptosis, however, is an active mode of cell death requiring the metabolism of cells. Thus, like thepermeability assays mentioned above, thecolorimetric assays may underestimate cellular damage and detect cell death only atthe later stages of apoptosis when the metabolic activity of the cells is reduced.Regardless of this disadvantage, the colorimetric assays are very useful for quantitating factor-induced cytotoxicity within a24 to 96 h period of cell culture.
However,these colorimetric assays are of limited value for measuring cell mediated cytotoxici-ty, since most effector cells become activated upon binding to the target cells. Thisactivation results in an increased formazanproduction by the effector cell, which tendsto mask the decreased formazan production resulting from target cell death.Note: Assays for cytotoxicity can be, andfrequently are, used to measure cell necrosis.1.3.2.1 Assays that measure plasmamembrane leakageWidely used standard methods for measuring plasma membrane leakage are based onthe uptake or exclusion of molecules withspecial light-absorbing or fluorescent properties.
Viable (intact plasma membrane)and dead (damaged plasma membrane) cellscan be discriminated by differential staining. Because dyes stain individual cells,each sample has to be analyzed by flow cytometry or microscopy. This kind of singlecell analysis is not suitable if many differentsamples have to be measured. In contrast,assays which quantitate plasma membranedisintegration in cell populations allowmany different samples to be handled simultaneously in a single MTP.in many cells and by the elaborate kineticassays required to quantitate most enzymeactivities.In contrast to the above mentioned cytoplasmic enzymes, lactate dehydrogenase(LDH) is a stable cytoplasmic enzyme present in all cells. It is rapidly released into thecell culture supernatant when the plasmamembrane is damaged. With the Cytotoxicity Detection Kit (see page 52), LDH activity can easily be measured in culture supernatants by a single point assay. The useof a spectrophotometric microplate reader(ELISA plate reader) allows the simultaneous measurement of multiple probes andthereby guarantees the easy processing of alarge number of samples (Figure 33).1LDH release assayIncubation(2 – 48 h)One group of standard assays performedin a MTP is based on the release of radioactive isotopes ([51Cr], [3H]-thymidine,[3H]-proline, [35S]- or [75Se]-methionine,5-[125I]-2-deoxy-uridine) or fluorescentdyes (bis-carboxyethyl-carboxyfluorescein(BCECF) or calcein-AM) from prelabeledtarget cells48, 49, 50.
The disadvantages ofsuch assays however, are (i) the use of radioactive isotopes in most of them, (ii) thenecessity for prelabeling of the target cells,and (iii) the high spontaneous release ofmost labels from the prelabeled target cells.Transfer ofsupernatantAnother group of assays is based on themeasurement of cytoplasmic enzymes released by damaged cells. The amount of enzyme activity detected in the culture supernatant corresponds to the proportion oflysed cells51, 52. Enzyme release assays havebeen described for alkaline and acid phosphatase, for glutamate-oxalacetate transaminase, for glutamate pyruvate transaminase, and for argininosuccinate lyase.However, their use has been hampered bythe low amount of those enzymes presentMeasurementCell Death – Apoptosis and NecrosisCytotoxicity Assay MethodsAssays that measure plasma membrane leakage+ reaction mixtureIncubation(0.5 – 4 h)ELISA-readerG Figure 33: Measurement of LDH activity ( ) usingthe microplate format (see also Flow Chart 14).51Cell Death – Apoptosis and NecrosisCytotoxicity Assay MethodsAssays that measure plasma membrane leakageCytotoxicity Detection Kit (LDH)Cat.
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