Apoptosis and Cell Proliferation (522915), страница 20
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Interestingly however, recent evidence suggests that mitochondrial electron transport may play a minorrole in the cellular reduction of MTT. Sincemost cellular reduction occurs in the cytoplasm and probably involves the pyridinenucleotide cofactors NADH and NADPH,the MTT assay can no longer be consideredstrictly a mitochondrial assay.]More recently, modified tetrazolium saltslike XTT62, 67, MTT68, and WST-1 (Figure45) have become available.
The major advantage of these new compounds is thatviable cells convert them to a water-solubleformazan. Thus, a metabolic assay with anyof these compounds requires one less step(solubilization of product) than an assaywith MTT. In addition, WST-1 is stableenough to be packaged as a ready-to-usesolution.Methods for studying cell proliferation and viability in cell populationsAssays that measure metabolic activityMTTFormazanNNNADHNNNHN+NNAD +CH 3NNNSCH 3SCH 3CH 3NO 2NO 2OONHFormazanSO–3H3CNADHNNNOSO–3N+NHNSO–3NAD +NHNSO–3ONO 2NOCell Proliferation and ViabilityCH 3XTTNO 2OCH 3CH 3NO 2WST-1N–FormazanNO 2O3SN2NADHNNHN+NNAD +SO–3NNISO–3SO–3IG Figure 45: Molecular structure of MTT, XTT, WST-1 and their corresponding reaction products.Since proliferating cells are metabolicallymore active than non-proliferating (resting) cells, the assays are suitable not onlyfor the determination of cell viability andfactor-mediated cytotoxicity (see Section1.3.2.2.) but also for the determination ofcell activation and proliferation.
However,one has to keep in mind that under nonideal cell culture conditions (such as the pHand D-glucose concentration in culturemedium), the MTT response may varygreatly in viable cells due to the metabolicstate of the cells (e.g., cellular concentrationof pyridine nucleotides)65, 69.These colorimetric assays are very rapidand convenient. Because this techniqueneeds no washing or harvesting of the cells,71Methods for studying cell proliferation and viability in cell populationsAssays that measure metabolic activitythe complete assay from the start of themicroculture to data analysis in an ELISAplate reader is performed in the same microtiterplate. In addition, an ELISA platereader linked with an on-line computer allows rapid and automated data processing(Figure 46).Cell Proliferation and ViabilityRoche Molecular Biochemicals offers threeMTP- based assays similar to the ones described in this section.
All three assays aresuitable for measurement of cell proliferation in response to growth factors, cytokines, mitogens and nutrients.Figure 46: Measurement of metabolicactivity using the tetrazolium salts MTT,XTT and WST-1. EOne of these assays uses MTT, whichforms an insoluble formazan product; theother two use tetrazolium salts (XTT andWST-1) that form soluble formazan products. All three assays are described on thefollowing pages.Note: For a more detailed discussion of theprinciples behind these metabolic assays, seethe topic, “Biochemical and cellular basis ofcell proliferation assays that use tetrazoliumsalts” (Appendix, page 113) in this guide.MTT-Assay+ MTTXTT-AssayWST-1-Assay+ XTT or WST-1Incubation(0.5 – 4 h)+ SolubilizationSolution2Incubation(1 – 24 h)MeasurementELISA-reader72ELISA-readerMethods for studying cell proliferation and viability in cell populationsAssays that measure metabolic activityCell Proliferation Kit I (MTT)2500 testsTypeColorimetric, MTP formatUseful forQuantitation of cell viability and proliferation as well as cytotoxicitySamplesAdherent or suspension cell culturesMethodIncubation of cells with MTT, followed by solubilization and spectrophotometric assay of colored productTime5–28 hTest principle: The assay is based on thereduction of the tetrazolium salt MTT byviable cells.
The reaction produces a waterinsoluble formazan salt which must be solubilized. The procedure involves:1Culturing the cells in a 96-well microtiterplate, then incubating them withMTT solution for approx. 4 h. Duringthis incubation period, viable cells convert MTT to a water-insoluble formazan dye.Kit contents1. MTT labeling reagent2. Solubilization solutionTypical results: See Figures 47 and 49.0.8Absorbance (A 550 nm – A 690 nm )Significance of kit: The Cell ProliferationKit I (MTT) measures the metabolic activity of viable cells. The assay is nonradioactive and can be performed entirely in amicrotiterplate (MTP).
It is suitable formeasuring cell proliferation, cell viabilityor cytotoxicity (see section 1.3 on page 50of this guide).Cell Proliferation and ViabilityCat. No. 1 465 0070.60.40.20.00.111010021000hIL– 6 [pg/ml ]23Solubilizing the formazan dye in theMTP.Quantitating the dye with an ELISAplate reader. The absorbance directlycorrelates with the cell number.Can be used to assay:P Adherent and suspension cells culturedin MTP.G Figure 47: Measurement of human Interleukin 6(hIL-6) activity on the mouse hybridoma cell line7TD1. Cells (2 x 103/well) were incubated in thepresence of various amounts of hIL-6.
After 4 days incubation, cell proliferation was analyzed by Cell Proliferation Kit I (MTT).Other applications: For more examples ofhow the Cell Proliferation Kit I (MTT) canbe used in the lab, see Appendix, page 125.73Methods for studying cell proliferation and viability in cell populationsAssays that measure metabolic activityCell Proliferation Kit II (XTT)Cat. No.
1 465 0152500 testsTypeColorimetric, MTP formatUseful forQuantitation of cell viability, proliferation, or cytotoxicitySamplesAdherent or suspension cell culturesMethodIncubation of cells with XTT, followed by spectrophotometric assay of colored productTime4hTest principle: The assay is based on the reduction of the tetrazolium salt XTT by viable cells in the presence of an electron coupling reagent. The reaction produces asoluble formazan salt. The procedure involves:1.11.00.9Absorbance (A 492 nm /A 690 nm )Cell Proliferation and ViabilitySignificance of kit: The Cell ProliferationKit II (XTT) measures the metabolic activity of viable cells.
The assay is nonradioactive and can be performed entirely in amicrotiterplate (MTP). It is suitable formeasuring cell proliferation, cell viability,or cytotoxicity (see section 1.3 on page 50of this guide).0.80.70.60.50.40.30.20.1122Culturing the cells in a 96-well microtiterplate, then incubating them withXTT solution for approx. 4 h. Duringthis incubation period, viable cells convert XTT to a water-soluble formazandye.Quantitating the formazan dye in theMTP with an ELISA plate reader.
Theabsorbance directly correlates with thecell number.Can be used to assay:P Adherent and suspension cells culturedin MTP.Kit contents1. XTT Labeling reagent2. Electron-coupling reagentNote: To prepare XTT labeling mixture,mix XTT labeling reagent with electroncoupling reagent prior to use.Typical results: See Figure 48.740.00.11101001000hTNF – a [pg/ml]G Figure 48: Measurement of human tumor necrosis factor a (hTNFa) activity on the mouse fibrosarcoma cell line WEHI-164.
After preincubation of thecells (1 x 106/ml) with actinomycin C (1 µg/ml) for 3 h,cells (5 x 104/well) were incubated in the presence of actinomycin C and various amounts of hTNFa for 24 h. Thecellular response was analyzed by Cell Proliferation Kit II(XTT).Other applications: For more examples ofhow the Cell Proliferation Kit II (XTT) canbe used in the lab, see Appendix, page 125.Methods for studying cell proliferation and viability in cell populationsAssays that measure metabolic activityCell Proliferation Reagent WST-12500 testsTypeColorimetric, MTP formatUseful forQuantitation of cell viability, proliferation, or cytotoxicitySamplesAdherent or suspension cell culturesMethodIncubation of cells with WST-1, followed by spectrophotometric assay ofcolored productTime0.5–4 hTest principle: The assay is based on the reduction of WST-1 by viable cells.
The reaction produces a soluble formazan salt. Theprocedure involves:12Culturing the cells in a 96-well microtiterplate, then incubating them withWST-1 for approx. 0.5–4 h. During thisincubation period, viable cells convertWST-1 to a water-soluble formazandye.Quantitating the formazan dye in theMTP with an ELISA plate reader.
Theabsorbance directly correlates with thecell number.3MTTXTTWST-12AbsorbanceSignificance of reagent: The Cell Proliferation Reagent WST-1 is a ready-to-usesubstrate which measures the metabolic activity of viable cells. The WST-1 assay isnonradioactive and can be performed entirely in a microtiterplate (MTP). It is suitable for measuring cell proliferation, cellviability or cytotoxicity (see section 1.3on page 50 of this guide).Cell Proliferation and ViabilityCat. No. 1 644 8071010 110 210 3cells/well10 410 5G Figure 49: Comparison of the sensitivity of various tetrazolium salts. P815 cells were preincubatedat various concentrations for 20 h before MTT (G), XTT(m) or Cell Proliferation Reagent WST-1 (P) was added.After 4 h substrate reaction, the absorbance was determined at the respective wavelength with an ELISA platereader.2For a detailed comparison of the WST-1 assay procedure with the MTT and XTT assays, see Flow Chart 17.Can be used to assay:P Adherent and suspension cells culturedin MTP.Typical results: See Figures 49–50.75Methods for studying cell proliferation and viability in cell populationsAssays that measure metabolic activityOther applications: For more examples ofhow the Cell Proliferation WST-1 can beused in the lab, see Appencix, page 125.1.00.60.50.3Absorbance (A 450 nm – A 690 nm )BrdU incorporation (A 370 nm – A 492 nm )0.9DNA synthesismetabolic activity0.41.63.16.2 12.5 25Actinomycin D (ng /ml)Cell Proliferation and Viability20.8G Figure 50: Combined use of the Cell ProliferationReagent WST-1 and the Cell Proliferation ELISA,BrdU (colorimetric) for the simultaneous measurement of cell viability and cell proliferation.
A549 cells(1 x 104/well in 100 µl) were incubated in the presenceof various amounts of actinomycin D for 20 h. Afterlabeling the cells with BrdU for 2 h, additionally CellProliferation Reagent WST-1 was added and cells werereincubated for another 2 h. Thereafter, the formazanformed was quantitated at 450 nm with an ELISA platereader (m). Subsequently, BrdU incorporation wasdetermined using the Cell Proliferation ELISA, BrdU(colorimetric) (P).Result: Actinomycin D inhibits DNA synthesis (P), but itdoes not inhibit the metabolic activity of the cell (m).Thus, actinomycin D is cytostatic (inhibition of DNAsynthesis) but not cytotoxic (no inhibition of metabolicactivity).Flow Chart 17: Assay procedures, CellProliferation Kit I (MTT), Cell ProliferationKit II (XTT), and Cell ProliferationReagent WST-1.
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