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Cell Counting Kit-8 (CCK-8) is a kit based on tetrazolium salt -8 (WST-8) assay. WST-8 assay is an optimized colorimetric method, which evaluates the cell viability, proliferation and cytotoxicity of mammalian cells according to their redox potential.

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Product Details of Cell Counting Kit-8 (CCK-8)

SMILES Code : NONE
MDL No. :N/A

Safety of Cell Counting Kit-8 (CCK-8)

GHS Pictogram:
Signal Word:Warning
Hazard Statements:H315-H319-H341-H351-H361-H373
Precautionary Statements:P201-P202-P260-P264-P280-P302+P352-P305+P351+P338-P308+P313-P332+P313-P337+P313-P362-P405-P501

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Description
Cell Counting Kit-8 (CCK-8) is a simple and highly sensitive tool commonly used to evaluate cell proliferation and cytotoxicity. The principle is colorimetric analysis based on water-soluble tetrazolium salt-8 (WST-8). In the metabolism of living cells, in the presence of electron carrier 1- methoxy -5- methylphenazine methyl sulfate (1-MethoxyPMS), dehydrogenase reduces tetrazolium salt WST-8 to formazan, which has the maximum light absorption at 450nm. This reaction occurs in cells with active metabolism, so it is considered as an indicator of cell vitality, and the color depth (the amount of formazan produced) has a linear relationship with the number of living cells in the sample.
Operating process
The kit can be used to detect cell viability, proliferation and cytotoxicity.
1) Making a standard curve.
a. Counting the number of cells in cell suspension with a cell counting plate, and then inoculating the cells into a 96-well plate;
b. Sequentially diluting with culture medium in proportion, generally making 5-7 cell concentration gradients, and and 4-6 repeats for each concentration are suggested.
c. After inoculation and culture for 4 hours, the cells adhere to the wall, and then 10μL CCK-8 reagent is added to 100μL of cell culture medium per hole, and the plate is gently shaken to make the generated dyes evenly distributed in the holes;
d. Incubate in a CO2 incubator at 37°C, and the incubation time varies from 30 minutes to overnight, depending on the cell type and cell concentration used. The OD value was measured at 450nm (430-490nm), and finally a standard curve with the number of cells as the abscissa (X axis) and the OD value as the ordinate (Y axis) was made. According to this standard curve, the cell number of unknown samples can be determined (the premise of using this standard curve is that the experimental conditions are completely consistent)
2) Cell proliferation and cytotoxicity test.
a. Determine the optimal cell numbers and incubation time of specific cells before the experiment;
b. Inoculate the cell suspension (100μL/ well) into a 96-well plate, and culture the cells in a CO2incubator at 37°C for 24 hours (toxicity test requires adding different concentrations of the drug to be tested in the culture plate and incubating for a period of time);
* If the drug to be tested is oxidizing or reducing, replace the fresh medium before adding CCK-8 to remove the influence of the drug to be tested. When the influence of the drug to be tested is relatively small, the culture medium cannot be changed, and the blank absorption after adding the drug to be tested in the culture medium can be directly deducted.
c. Add 10μL CCK8 reagent to each well (no bubbles), gently shake the plate to make the dye evenly distributed in the wells, and the incubation time in the cell incubator ranges from 30 minutes to overnight;
d. The absorbance at 450nm(430-490nm) was measured by enzyme-labeled instrument. e. Calculating the survival rate or inhibition rate of cells.
Cell survival rate %= [A (dosing) -A (blank)]/ [A (0 dosing) -A (blank)]x100%
Inhibition rate %= [A (0 dosing) -A (dosing)]/ [A (0 dosing) -A (blank)] X100%
A (dosing): The absorbance with cells, CCK-solution and drug solution pores.
A (blank): The absorbance with culture medium and CCK-solution without cell pores.
A (0 dosing): absorbance with cells and CCK-solution but without drug solution pores.
Cell survival rate% and inhibition rate% showed cell proliferation activity or cytotoxicity to some extent.
 

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