EnoGeneCell™ Counting Kit-8 (CCK-8)
Cell Counting Kit-8 (CCK-8) allows very convenient assays by utilizing 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 a water-soluble formazan dye upon reduction in the presence of an electron mediator. Cell Counting Kit-8 is a one-bottle solution; no premixing of components is required. Cell Counting Kit- 8, being nonradioactive, allows sensitive colorimetric assays for the determination of the number of viable cells in cell proliferation and cytotoxicity assays. WST-8 is reduced by dehydrogenases in cells to give a yellow colored product (formazan), which is soluble in the tissue culture medium. The amount of the formazan dye generated by the activity of dehydrogenases in cells is directly proportional to the number of living cells. The detection sensitivity of CCK-8 is higher than other tetrazolium salts such as MTT, XTT, MTS or WST-1.
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Description
Cell Counting Kit-8 (CCK-8) allows very convenient assays by utilizing 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 a water-soluble formazan dye upon reduction in the presence of an electron mediator. Cell Counting Kit-8 is a one-bottle solution; no premixing of components is required. Cell Counting Kit- 8, being nonradioactive, allows sensitive colorimetric assays for the determination of the number of viable cells in cell proliferation and cytotoxicity assays. WST-8 is reduced by dehydrogenases in cells to give a yellow colored product (formazan), which is soluble in the tissue culture medium. The amount of the formazan dye generated by the activity of dehydrogenases in cells is directly proportional to the number of living cells. The detection sensitivity of CCK-8 is higher than other tetrazolium salts such as MTT, XTT, MTS or WST-1.
Product Information
Cell Proliferation Assay
1) Inoculate cell suspension (100 μl/well) in a 96-well plate. Also prepare wells that contain known numbers of viable cells (to create a calibration curve in step 4). Pre-incubate the plate in a humidified incubator (e.g., at 37°C, 5% CO2).
2) Add 10 μl of the CCK-8 solution to each well of the plate.
Be careful not to introduce bubbles to the wells, since they interfere with the O.D. reading.
3) Incubate the plate for 1-4 hours in the incubator.
4) Measure the absorbance at 450 nm using a microplate reader. Prepare a calibration curve
using the data obtained from the wells that contain known numbers of viable cells.
To measure the absorbance later, add 10 μl of 1% w/v SDS to each well, cover the plate and store it with
protection from light at room temperature. No absorbance change should be observed for 48 hours.
Cytotoxicity Assay
1) Dispense 100 μl of cell suspension (5000 cells/well) in a 96-well plate.
2) Pre-incubate the plate for 24 hours in a humidified incubator (e.g., at 37°C, 5% CO2).
3) Add 10 μl of various concentrations of toxicant into the culture media in the plate.
4) Incubate the plate for an appropriate length of time (e.g., 6, 12, 24 or 48 hours) in the incubator.
5) Add 10 μl of CCK-8 solution to each well of the plate.
Be careful not to introduce bubbles to the wells, since they interfere with the O.D. reading.
6) Incubate the plate for 1-4 hours in the incubator. Measure the absorbance at 450 nm using a microplate reader.
To measure the absorbance later, add 10 μl of 1% w/v SDS to each well, cover the plate and store it with
protection from light at room temperature. No absorbance change should be observed for 48 hours.
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