Assay Principle

Enhanced Cell Counting Kit 8 (CCK8), short for WST-8, is a rapid, high-sensitivity test kit based on WST-8 for cell viability, proliferation and cytotoxicity. This kit takes only 0.5 to 1 hour to complete the test, and is faster, more sensitive, and has a wider linear range than conventional or enhanced CCK-8 kits. This kit is suitable for determination of absorbance at around 450nm.

WST-8 is a compound similar to MTT that, in the presence of electron-coupled reagents, can be reduced by some dehydrogenase enzymes within the mitochondria to produce orange-yellow formazan. The more cells proliferate and the faster, the darker the color; The more cytotoxic, the lighter the color. There is a linear relationship between the depth of color and the number of cells. Compared with WST-1, WST-8 is more sensitive, more soluble, and more stable. This kit has been optimized to greatly reduce incubation time, generally only 0.5-1 hours to complete the test.

The test kit is very convenient. There is only one tube of enhanced CCK-8 solution that has been prepared, and no further preparation and other operations are required. Without the use of isotopes, all detection steps are performed in the same 96-well plate. It can be used for the detection of large quantities of samples.

Phenol red and serum had no significant effect on the determination of this kit. This product has no obvious toxicity to cells. After adding enhanced CCK-8 solution for color development, it can be repeatedly read with the enzyme marker at different times, making the detection time more flexible and convenient to find the best determination time. With a 96-well plate requiring 10μl enhanced CCK-8 solution per 100μl cell, this kit can perform 100 tests per 1ml.

Assay Procedure

1. Usually 2000 cells with 100 microliters are added to each well for cell proliferation experiments, and 5000 cells with 100 microliters are added to each well for cytotoxicity experiments (the number of cells used in each well depends on the cell culture days, the speed of cell proliferation and other factors). At the same time, a culture medium hole without cells was set up as a negative control, and the cells were cultured according to the cell culture program. If necessary, drug treatment cells can be added.

2. Add 10 microliters of enhanced CCK-8 solution per well. If the initial culture volume is 200 microliters, 20 microliters of enhanced CCK-8 solution should be added, and so on in other cases. If there is concern that the drug used will interfere with the detection, a blank control should be set up in which the appropriate amount of cell culture solution, drug and enhanced CCK-8 solution are added but no cells are added.

3. Continue to incubate in the cell incubator for 0.5-3 hours, for most cases, incubation for 0.5-1 hours is fine.The length of time depends on the type of cells and the density of cells and other experimental conditions. In the first experiment, the enzyme labeling instrument can be used to detect 0.5, 1, 2 and 3 hours later, respectively, and then a time point with a more appropriate absorbance range is selected for subsequent experiments.

4. Measurement of absorbance at 450nm. If no 450nm filter, 420-480nm filter can be used.