Catalog No.: CSK1160Kit ContentCCK-8 solution 5mlStorageStore at 4℃ in dark for oneyearIntroductionCell Counting Kit-8 (CCK-8) allows very convenient assays byutilizing Dojindo’s highly water-soluble tetrazolium salt. WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2H-tetrazolium, monosodium salt]* produces awater-soluble formazan Dye upon reduction in the presence of anelectron carrier,as shown in Figure 1.Cell Counting Kit-8 is a onebottle solution; no premixing of components is required. CellCounting Kit-8, being nonradioactive, allow sensitive colorimetricassays for the determination of the number of viable cells n cellproliferation and cytotoxicity assays. WST-8 is educed bydehydrogenases in cells to give a yellowcolored roduct (formazan),which is soluble in the tissue culture medium. The amount of theformazan dye generated by the activity of dehydrogenases in cellsis directly proportional to the number of living cells. The detectionsensitivity of CCK-8 is higher than other tetrazolium salts such asMTT, XTT, MTS or WST-1.The kit components are sufficient for performing up to 500assays.
Protocol1. Collect logarithmic phase cell, adjust cell suspension concentration; add 100ul floor plate. In general,cells seeded at densities between 1000-10,000 cells per well (side holes filled with aseptic PBSbuffer).2. Seed cells in a 5% Co2 incubator at 37℃ until cells bespread well bottom for one floor (cells numberfor each well is according to cells’ size and breed speed). Add concentration gradient drug.Principlely, add drug after cells adhere. 0-10ul per well. Using 3-5 repeating pipettors.3. Add 10μl CCK-8 into each well.( considering the ratio 1:10). Choose the wells without cells ascontrast wells.4. Incubate for 0.5-4 hours, usually 1 hour is enough. The incubate time response to the situation ofcell’s type and concentration. You can try to read the result after 0.5 hour, 1hour, 2 hours and 4hours solely at first time, then chose a proper time for next step.5. Read absorbance at 450nm. If there’s no 450nm filter, use 420-480nm instead. During dualwavelength spectrophotometry, you may choose wavelength longer than 600nm.Note1. If cell culture time is too long, please pay attention to the evaporation issue. Avoid using the outmostwells, add PBS buffer, water or culture fluid instead; or place 96 wells near by the water in incubator.2. This kit bases on the catalytic reaction of dehydrogenates. If there are too many reducing agents inthe system ready to detect, such as some antioxidant which may interrupt the result, you shouldremove them first.3. Make sure that there’s no bubble in any well before use the ELISA reader, or it will interrupt theresult.4. Please wear transparent gloves when operate.