Apoptosis and Cell Proliferation (522915), страница 22
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No. 1 669 9152Figure 55: How the Cell ProliferationELISA, BrdU (colorimetric) works. HFixDenatOne-step fixation of cells and denaturationof BrdU-labeled DNAAnti-BrdU-PODfab fragmentsTMBsubstrate81Methods for studying cell proliferation and viability in cell populationsAssays that measure DNA synthesisLabel cells with BrdU (2–18 h, 37°C)Suspension cellsAdherent cellsRemove labeling medium Remove labeling mediumusing a needleby inverting the MTPAir dry cells (10 min,hairdryer)Can be used to assay:Add FixDenat solution and incubate (30 min, RT)P Adherent cells as well as cells in suspension cultured in 96-well microtiterplates(e.g. cell lines, activated peripheralblood lymphocytes and other in vitroproliferating cells).Cell Proliferation and ViabilityTap MTP to remove FixDenat then add Anti-BrdU-PODand incubate (30–90 min, RT)Wash MTP 3 times (15 min, RT)Add substrate and incubate (5–20 min, RT)Cell Proliferation ELISA, Cell Proliferation ELISA,BrdU (colorimetric)BrdU (chemiluminescence)Measure absorbanceusing an ELISA platereader (2 min, RT)Measure light emissionusing a luminometer(2 min, RT)G Flow Chart 19: Assay procedures, Cell ProliferationELISA, BrdU (colorimetric) and Cell Proliferation ELISA,BrdU (chemiluminescence).Sensitivity: The Cell Proliferation ELISABrdU (colorimetric) and Cell ProliferationELISA, BrdU (chemiluminescence) are assensitive as the [3H]-thymidine-based cellproliferation assay.2Note: The ability to detect a minimumnumber of proliferating cells in a certainsample depends on the amount of BrdUincorporated into the cells and thus on thelabeling period.
In most cases, detectionrequires a labeling period of 2 to 24 h.The use of a chemiluminescence substrateallows the measurement of cell proliferation over a broad range. This range is directly comparable to the measuring range ofthe [3H]-thymidine-based cell proliferationassay.82Specificity: The antibody conjugate (antiBrdU-POD, Fab fragments) will bind toBrdU-labeled DNA after the DNA is denatured.
The antibody specifically recognizes 5-bromo-2’-deoxyuridine; it showsno cross-reactivity with any endogenouscellular components such as thymidine oruridine.Kit contentsCell Proliferation ELISA, BrdU(colorimetric):1. BrdU labeling reagent (1000 x), sterile2. Anti-BrdU-POD Fab fragments3. Antibody dilution solution (ready-touse)4. Washing buffer (10 x)5. FixDenat (ready-to-use)6. TMB-substrate solution (ready-to-use)Cell Proliferation ELISA, BrdU(chemiluminescence):1. BrdU labeling reagent (1000 x), sterile2.
Anti-BrdU-POD Fab fragments3. Antibody dilution solution (ready-touse)4. Washing buffer (10 x)5. FixDenat (ready-to-use)6. Substrate component A (luminol/4-iodophenol)7. Substrate component B (peroxide)Note: The FixDenat solution (included inthe kits) is also available as a separate reagent (Cat. No. 1 758 764, 4 x 100 ml [enoughfor 2000 tests]).
This ready-to-use solutionsimplifies detection of BrdU-labeled DNAin ELISA applications, since it simultaneously fixes cells and denatures DNA to expose BrdU epitopes.Typical results: See Figures 56–59.Methods for studying cell proliferation and viability in cell populations2.5100.510 210 3cells/well10 4010 5G Figure 56: Comparison of the sensitivity of theCell Proliferation ELISA, BrdU (colorimetric) and theradioactive thymidine incorporation assay formeasuring proliferation in various concentrations ofcells. Various concentrations of L929 cells were culturedin the wells of a microtiter plate.
Duplicate cultures ofeach cell concentration were labeled for 4 h with eitherbromodeoxyuridine (BrdU) or tritiated thymidine ([3H]TdR). The cells were assayed for cell proliferation witheither the Cell Proliferation ELISA, BrdU (BrdU labeling,P) or a standard filtration/liquid scintillation countingprotocol ([3H]-TdR labeling, p).Result: The Cell Proliferation ELISA, BrdU (colorimetric)measures proliferation with a sensitivity comparableto the radioactive thymidine assay at all cell concentrations.1.4251.2201.00.8150.6100.450.2010 -310 -210 -110 0PHA [mg/ml]010 1G Figure 57: Comparison of the Cell ProliferationELISA, BrdU (colorimetric) and the radioactivethymidine incorporation assay for measuring stimulation of various concentrations of mitogen.
Humanperipheral blood lymphocytes were cultured in thepresence of varying concentrations of phytohemagglutinin (PHA) in the wells of a microtiter plate. Duplicatecultures from each PHA concentration were labeled for4 h with either bromodeoxyuridine (BrdU) or tritiatedthymidine ([3H]-TdR). The cells were assayed for cellproliferation with either the Cell Proliferation ELISA, BrdU(BrdU labeling, P) or a standard filtration/liquid scintillation counting protocol ([3H]-TdR labeling, p).Result: The Cell Proliferation ELISA, BrdU (colorimetric)is able to detect mitogen-stimulation with a sensitivitycomparable to the radioactive thymidine assay.2.01.51.00.5control 1 µg/ml 1 ng/ml 10 µg/ml10 µg/ml10 µg/ml0.001 %PHA OKT3 ConA PWM SAC10610BrdU[ 3 H]-TdR6101054101043103[ 3 H]-thymidine incorporation (cpm )1.0BrdU incorporation (A 405 nm – A 490 nm )20patienthealthyvolunteerrelative light units (rlu/s)1.501012.5302.0F Figure 58: Reduced PBL proliferation of an immunosuppressedpatient in response to variousmitogens.
Cells (1 x 105/well) froma healthy volunteer (m) or an immunosuppressed patient ( m) wereincubated in the presence of variousmitogens for 56 h. Cells were labeledwith BrdU for 16 h, then cell proliferation was analyzed by Cell ProliferationELISA, BrdU (colorimetric).
The errorbars indicate the maximum and minimum values of triplicate microcultures(data from T. Brüning, [1994] Klin. Lab.40, 917–927, Figure 3). Mitogens usedwere: PHA (phytohemagglutinin), OKT3(anti-CD3 monoclonal antibody), Con A(concanavalin A), PWM (pokeweedmitogen), and SAC (Staphylococcusaureas Cowan I).Result: The BrdU ELISA clearly detectedthe difference in response betweenthe healthy and immunosuppressedsubjects.10MediumINV-KAINV-AINV-BRUVHSV2G Figure 59: Measurement of the proliferation of antigen-activated PBL.
Cells (1 x 105/well)were incubated in the presence of various viral antigens on culture medium alone for 5 days. Afterlabeling with BrdU ( m) or [3H]-TdR (m) for 16 h, cell proliferation was analyzed by Cell ProliferationELISA BrdU (chemiluminescence) ( m) or LSC (m). Antigens used were: INV-KA (influenza controlantigen), INV-A (influenza virus A), INV-B, (nfluenza virus B), RUV (Rubella virus), and HSV (herpes simplex virus type I).Result: The Cell Proliferation ELISA, BrdU (chemiluminescence) detected antigen stimulation with asensitivity comparable to the radioactive thymidine assay.Other applications: For more example ofhow the Cell Proliferation ELISAs can beused in the laboratory, see Appendix, page125.83Cell Proliferation and ViabilityAssays that measure DNA synthesis2Methods for studying cell proliferation and viability in all populationsMethods for studying cell proliferation and viability in all populationsSummary of methods for studying cell proliferation and cell viability in cell populationsSummary of methods for studying cell proliferation and cell viability in cell populations2.2.1.3 Summary of methods for studying cell proliferation and cell viability in cell populationsDNA SynthesisLabelAssay PrincipleAdvantagesLimitations[3H]-TdR Proliferation Assay[3H]-TdRP [3H]-TdR is added to cells cultured in MTP and the cells are incubated (usually forP Sensitive (103–104 cells/test required)P Linear measurement of cell proliferation over a broad, logarithmicP Radioactive isotope handling and storage problemsP Long half lifeP Radioactive waste disposal costs2–24 h).
During this labeling period, [3H]-TdR is incorporated into the DNA ofproliferating cells.P Cells are harvested by vacuum aspiration onto glass fiber filters. While free [3H]-TdRis washed through the filters, the [3H]-TdR incorporated in the DNA is retained.P The radioactivity retained on the filters is measured by liquid scintillation counting(LSC).BrdU incorporation assayP BrdU is added to cells cultured in MTP and the cells are incubated (usually forBrdU2–24 h).
During this labeling period BrdU is incorporated into the DNA of proliferatingcells.P After the culture supernatant is removed, the cells are fixed and then incubated withan anti-BrdU antibody conjugated with peroxidase (anti-BrdU-POD).P This antibody binds to BrdU which has been incorporated into the DNA.P Bound anti-BrdU-POD is detected by a substrate reaction and quantified by an ELISAplate reader.Cell Proliferation and ViabilityBrdU Labeling and Detection Kit III2BrdU incorporation assayP No transfer of the cells; the entire assay is performed in a single MTP P Assay is not linear over a broad logarithmic range of cell proliferation(limitation of the ELISA plate reader)P Non-radioactiveP 3 washing and incubation stepsP Longer assay timepage 79of this guidepage 81of this guideBrdUSee above (BrdU incorporation assay)P No transfer of the cells; the entire assay is performed in a single MTP P Assay is not linear over a broad logarithmic range of cell proliferation(limitation of the ELISA plate reader)P 1 washing and 2 incubation steps onlyP Short assay timeP Robust system: low standard deviationP Sensitive (103–104 cells/test required)BrdUSee above (BrdU incorporation assay)P [see also Cell Proliferation ELISA, BrdU (colorimetric)]P Linear measurement of cell proliferation over a broad, logarithmicCell Proliferation ELISA, BrdU (colorimetric)BrdU incorporation assayrangeP Low backgroundCell Proliferation ELISA, BrdU (chemiluminescence)For productinformation, seeP For chemiluminescence measurement special MTP (Black with clear, page 81flat bottom) requiredof this guiderangeG Table 16: Summary of methods to study DNA synthesis in cell populations.Metabolic activityMethod/Roche Molecular Biochemicals productLabelAssay PrincipleAdvantagesMTT Assay61Non-isotopicP MTT solution is added to cells cultured in MTP and the cells are incubated (usually forP No transfer of the cells; the entire assay is performed in a single MTP P Assay is not linear over a broad logarithmic range of cell proliferationdue to the ELISA plate readerP MTT is metabolized by all cells; the assay can be used with all celltypesP Insoluble reaction product; resolubilization of the reaction productrequiredP InexpensiveP Connot take multiple time points in a single assayP Cells with low metabolic activity (e.g., lymphocytes) must be used in4 h).
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