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Jason Little - Dojindo Molecular Technologies Technical Assistance
Background of Cell Counting Kit-8
Cell Counting Kit-8 (CCK-8) allows convenient assays using Dojindo's tetrazolium salt, WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt), which produces a water-soluble formazan dye upon bioreduction in the presence of an electron carrier, 1-Methoxy PMS. CCK-8 solution is added directly to the cells; no pre-mixing of components is required. CCK-8 is a sensitive nonradioactive colorimetric assay for determining the number of viable cells in cell proliferation and cytotoxicity assays. WST-8 is bioreduced by cellular dehydrogenases to an orange formazan product that is soluble in tissue culture medium. The amount of formazan produced is directly proportional to the number of living cells. The detection sensitivity of cell proliferation assays using WST-8 is higher than assays using the other tetrazolium salts such as MTT, XTT, MTS or WST-1.
<ref>TReview - "Why is the water-soluble formazan necessary?" is available</ref>
Materials Needed for Cell Counting Kit-8
Microplate reader: 450-490 nm filter
96 well microplate
Multipippet (8 or 12 channel for 10 - 100 ul)
Cell counter or Hematocytometer
Procedure of Cell Counting Kit-8
1. Prepare a cell suspension with 50,000-100,000 cells/ml using an appropriate culture medium.
2. Add 100 Âµl of the cell suspension to each well of a 96-well plate.
3. Pre-incubate the plate at 37 ÂºC.a)
4. Add 10 Âµl of various concentrations of a solution to be testedb) to each well.
5. Incubate the plate at 37 ÂºC for a certain time period, such as 24, 48 or 72 hours.
6. Add 10 Âµl CCK-8 solutionc) to each well, and incubate the plate at 37 ÂºC for 1-4 hours.d)
7. Read the O.D. at 450 nm to determine the cell viability in each well.
a) Overnight preincubation in a CO2 incubator is recommended.
b) Use the culture medium or PBS to prepare the solutions.
c) If the solution to be tested has reducing activities, incubate the solution and CCK-8 without cells to determine the background absorbance at 450 nm. If the absorbance is negligibly small, add CCK-8 solution to each well. If the absorbance is high, remove the culture medium and wash cells twice with the medium, then add 100 Âµl of the culture medium and 10 Âµl CCK-8 solution.
d) Longer incubation may be necessary for leukocyte cells.
Cell Counting Kit-8 Notes
References for Cell Counting Kit-8
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