Apoptosis and Cell Proliferation (522915), страница 16
Текст из файла (страница 16)
No. 1 644 79312000 testsTypeColorimetric assay, MTP formatUseful forQuantitation of LDH activity released from damaged/dying cellsSamplesCell-free supernatants from cells in cultureMethodPreparation of cell-free supernatant, followed by incubation of supernatantwith INT to form colored formazan, a product which may be quantitatedcolorimetricallyTime0.5–1h (+ induction of cell death)Significance of kit: The Cytotoxicity Detection Kit measures cytotoxicity and celllysis by detecting LDH activity releasedfrom damaged cells. The assay is performedin a 96-well MTP. The kit can be used inmany different in vitro cell systems wheredamage of the plasma membrane occurs.Examples are:P Detection and quantification of cell mediated cytotoxicity.1Incubating the cells in culture to allowcell death to occur. An increase in theamount of dead or plasma membranedamaged cells during the assay results inan increase of LDH in the culture supernatant.2Collecting the cell-free culture supernatant.3Adding the substrate mixture from thekit to the culture supernatant.
AnyLDH released into the supernatant during Step 1 will reduce the tetrazoliumsalt INT to formazan by a coupled enzymatic reaction. Thus, release of LDHinto the supernatant directly correlatesto the amount of formazan formed inthis step.4Quantitating the formazan dye formedin an ELISA plate reader. The formazandye formed is water-soluble and showsa broad absorption maximum at about500 nm.P Determination of mediator-induced cytolysis.P Determination of the cytotoxic potential of compounds in environmental andmedical research, and in the food, cosmetic, and pharmaceutical industries.Figure 34: Biochemistry of the Cytotoxicity Detection Kit (LDH): In the firstenzymatic reaction LDH reduces NAD+to NADH + H+ by oxidation of lactate topyruvate; in the second enzymatic reaction the catalyst (diaphorase) transfersH/H+ from NADH + H+ to the tetrazolium salt INT.HP Determination of cell death in bioreactors.Test principle: The assay is based on a thecleavage of a tetrazolium salt when LDH ispresent in the culture supernatant.
The procedure involves:For a detailed overview of the steps involved in the procedure, see Figures 33 and34 and Flow Chart 14.1st enzymatic reactionpyruvatelactateCOO –COO –LDHCOHOCH 3NCH 3NADH + H +2 nd enzymatic reactionNAD +NNCl –CNHCN+HNCl –CCatalystNN+HNO 2tetrazolium salt (pale yellow)52NO 2formazan salt (red)Incubate cells in a MTP or batch culture for a certainperiod of time (optional: induction of death by treatment with compounds or test substances)Remove 100 ml supernatant and transfer it to anotherMTPAdd 100 µl/well reaction mixture to each supernatantand incubate for 0.5 hE/T cellE/T-controlControlAbsorbance (A 492 nm /A 690 nm )Centrifuge cellsF Figure 35.
Determination of thecytolytic activity of allogen-stimulated cytotoxic T lymphocytes (CTLs)using the Cytotoxicity Detection Kit(LDH). Spleen cells of C57/BI 6 mice(H-2b) were stimulated in vitro withP815 cells (H-2d). Viable CTLs werepurified and titrated in the microplate asdescribed in the package insert. Targetcells (1 x 104 cells/well) were incubatedin the presence or absence (effector cellcontrols) of effector cells for 4 hours.Culture supernatant samples (100 µl/well) for effector controls and the effector-target cell mix were assayed forLDH activity.
The middle curve is generated when the background controlvalues are subtracted from the effectortarget cell values.1.51.00.5Measure absorbance using an ELISA plate readerG Flow Chart 14: Assay procedure, Cytotoxicity Detection Kit (LDH).0110100effector:target cell ratioCan be used to assay:Typical results: See Figures 35 and 36.2.5viable cellsdead cellsLDH activity2.015Cells/ml [u 10 -5 ]Kit contents1. Catalyst (Diaphorase/NAD + mixture)2.
Dye solution (INT and sodium lactate)Note: To prepare the reaction mixture,mix catalyst with dye solution prior touse. Purified LDH (Cat. No. 107 077)may be used as a positive control.201.5101.05Absorbance (A 492 nm – A 690 nm )P Cell-free supernatants obtained fromcells cultured in 96-well microplates orbatch cultures.F Figure 36: Correlation of celldeath (defined by increased plasmamembrane permeability) and LDH release.
Ag8 cells (starting cell concentration: 2 x 105 /ml) were cultured andafter 1, 2, 3 and 5 days, aliquots wereremoved. The amount of viable (G) anddead (m) cells was determined bytrypan blue exclusion. LDH activity incell free culture supernatant was determined using the Cytotoxicity DetectionKit (v).Result: Increased LDH release clearlycorrelated with the increase of deadcells.0.500.00123456Days in cultureOther applications: For more examplesof how the Cytotoxicity Detection Kit(LDH) can be used in the lab, see Appendix, page 122.53Cell Death – Apoptosis and NecrosisCytotoxicity Assay MethodsAssays that measure plasma membrane leakage1Cell Death – Apoptosis and NecrosisCytotoxicity Assay MethodsAssays that measure plasma membrane leakageCellular DNA Fragmentation ELISACat.
No. 1 585 0451500 testsTypeSandwich ELISA, colorimetricUseful forQuantitation of BrdU-labeled DNA fragments either released from cellsduring necrosis or cell-mediated cytotoxicity, or within the cytoplasm of apoptotic cellsSamplesCell-free supernatants from cultured cells or cytoplasmic lysates of cells, prelabeled with BrdUMethodPrelabeling of cells with BrdU, followed by immunodetection of BrdU-labeled DNA fragments in sampleTime4.5–5.5 h (+ BrdU labeling and induction of cell death)Significance of kit: The Cellular DNAFragmentation ELISA measures apoptosis,necrosis, or cell mediated cytotoxicity byquantitating the fragmentation and/orrelease of BrdU-labeled DNA. The kit detects DNA fragments:1Prelabeling of cells with BrdU.2Incubating the labeled cells in thepresence of either an apoptosis inducingagent or effector cells (for cell mediatedcytotoxicity).
At the end of the incubation, cells are centrifuged and either supernatant is analyzed (for cell mediatedcytotoxicity or necrosis) or cellular lysate is analyzed for apoptosis. The supernatant, containing LMW-DNA isused for the assay. If desired, both sample types can be prepared and assayed(See Flow Chart 15).3Adsorbing the Anti-DNA antibodyonto the wells of a MTP.4Adding the supernatant of Step 2 to theMTP.
BrdU-labeled DNA fragmentsin the sample bind to the immobilizedAnti-DNA antibody.5Denaturing the immunocomplexedBrdU-labeled DNA-fragments by microwave irradiation or nuclease treatment. This procedure is necessary forthe accessibility of the BrdU antigen.6Reacting Anti-BrdU antibody peroxidase conjugate (Anti-BrdU-POD) withthe BrdU-labeled DNA to form an immunocomplex.7Quantitating the bound Anti-BrdUPOD in the immunocomplex with aperoxidase substrate (TMB).P In the cytoplasm of apoptotic cells, thusproviding a non-radioactive alternativeto the [3H]-thymidine-based DNAfragmentation assay.P Released into the culture supernatantduring cell mediated cytotoxicity, thusproviding a non-radioactive alternativeto the [3H]-thymidine- and [51Cr]-release assays.Test principle: The assay is a sandwichenzyme-linked immunosorbent assay(ELISA).
It uses two mouse monoclonalantibodies: one directed against DNA theother against BrdU (Figure 37). The procedure (Flow Chart 15) involves:Figure 37: How the Cellular DNAFragmentation ELISA works.HmicrowaveAnti-DNA-coated MTP54BrdU-labeledDNA-fragmentsAnti-BrdU-PODfab fragmentsTMBsubstrateSample preparation:Label cells in tissue culture flask with BrdU (2–18 h, 37°C)F Flow Chart 15: Assay procedure,Cellular DNA Fragmentation ELISA.Aliquot BrdU-labeled cells into the MTPMeasurement of apoptosisMeasurement of cell mediated cytotoxicityor necrosisAdd cell death-inducing agentAdd effector cellsCell Death – Apoptosis and NecrosisCytotoxicity Assay MethodsAssays that measure plasma membrane leakage1Incubate cells (1–24 h, 37°C)Add lysis buffer and incubate (30 min, RT)Centrifuge MTP (10 min, 250 x g )Remove supernatant and analyze by ELISAELISACoat MTP with Anti-DNA antibody (1 h, RT)Recoat microtiter plate with blocking solution (30 min, RT)Wash MTP, then add supernatant and incubate (90 min, RT)Option 1Option 2Wash MTP, then denature/fix sample by microwaveradiation (5 min)Wash MTP, then add nuclease and incubate(30 min, 37°C)Wash MTPAdd Anti-BrdU-POD and incubate (90 min, RT)Wash MTP, then add substrate and incubate (10–30 min, RT)Measure absorbance using an ELISA plate reader (2 min, RT)55Cell Death – Apoptosis and NecrosisCytotoxicity Assay MethodsAssays that measure plasma membrane leakageFlow Chart 16: Simultaneous analysisof apoptosis and necrosis in thesame sample with the Cellular DNAFragmentation ELISA.
ELabel cells with BrdUAdd cell death-inducing agentSplit sample into two partsCentrifuge cellsRemove supernatant totallyTransfer 100 ml supernatant into Anti-DNAprecoated MTPLyse cellsAssay supernatant for BrdU-labeled DNACentrifuge lysate (to pellet HMW DNA)Result: Measurement of necrosis1Assay supernatant for oligonucleosomes containingBrdU-labeled LMW DNAResult: Measurement of apoptosisSensitivityApoptosis: When HL60/CAM is used as amodel system for apoptosis, the ELISA candetect BrdU-labeled DNA fragments in thecytoplasm of 1 x 103 cells/well (Figure 36).Cell mediated cytotoxicity: When allogeneic-stimulated cytotoxic T cells are usedas effector cells to lyse P815 target cellsin a cell mediated cytotoxicity assay, theELISA can detect BrdU-labeled DNAfragments from 2 x 103 target cells/well.Note: The ability to detect a minimumnumber of dying/dead cells in a particularsample strongly depends on the kinetics ofcell death, the cytotoxic agent or the effectorcells used to induce cell death, and theamount of BrdU incorporated into the target cells.SpecificityE The Anti-DNA antibody binds tosingle-and double-stranded DNA.
![](https://s.studizba.com/z.php?f=/templates/si/i/noavatar.png&w=100&h=100&t=1"){kind=avatar}
