What is the CCK-8 assay?

What is CCK-8?

Cell Counting Kit-8 (CCK-8) is a readily available, easy-to-use cell viability and cytotoxicity assay. CCK-8 takes advantage of WST-8 [2- (2- methoxy-4-nitrophenyl)-3- (4-nitrophenyl)-5- (2, 4-disulfophenyl)-2H- tetrazolium, monosodium salt] to directly measure cell viability. Specifically, WST-8 is a water-soluble salt that can be reduced by dehydrogenases in viable cells to a water-soluble formazan dye. The biochemical reaction causes a change in color that can be easily quantified by measuring absorbance at 450nm. This simple and convenient colorimetric assay has been widely used to determine cell viability and cytotoxicity of various agents in cell culture. Boster Bio offers an affordable, highly cited Cell Counting Kit-8 (CCK-8) (Catalog# AR1160) for use in your cell culture experiments. CCK-8 assays are quick and easy to use since they do not require thawing or mixing any reagents before use. Simply add the reagent to cells in culture, incubate cells at standard conditions for up to four hours, then determine cell viability or cytotoxicity by measuring the colorimetric change in a cell plate reader at 450 nm.

What does CCK-8 measure?

CCK-8 measures cell viability by correlating the production of colored formazan dye to the amount of living cells in culture. Dehydrogenases from viable cells convert WST-8 into a formazan dye, which produces an easily measurable color change that can be quantified in a cell plate reader at 450 nm.

What is the difference between cell viability and cytotoxicity?

Cell viability assays often measure the amount of living, healthy cells while cytotoxicity assays typically measure the amount of dead or apoptotic cells in culture. The CCK-8 assay has been used in both types of assays though it is measuring the number of living cells. The cell counting kit-8 can be used in combination with common or novel drugs to test cytotoxicity by measuring the number of viable cells remaining after treatment with different concentrations of drugs. One of the advantages of the CCK-8 assay is that WST-8 is not toxic to cells, so it can be used in combination with potentially toxic reagents to measure cytotoxicity.

What is the best cell viability assay?

There are numerous cell viability assays, such as CCK-8, MTT, and ATP-cell viability assays, each having its own advantages and disadvantages. MTT assays are most similar to CCK-8 assays since MTT is catalyzed into a purple-colored formazan that can be measured in a cell plate reader. However, MTT assays are more inconvenient than CCK-8 assays, since MTT is not water-soluble, must be thawed prior to use, and must be made dissolvable in the cell culture. On the other hand, ATP-cell viability assays utilize ATP produced from healthy, viable cells and luciferase enzyme added in vitro to convert the luciferin reagent into a luminescent product that can be measured in a cell plate reader. While this is also a relatively simple and quick procedure that measures luminescence instead of color change, ATP-cell viability assays tend to be more expensive than other methods like CCK-8. CCK-8 offers better sensitivity to cell viability than other assays as well. While the best cell viability assay will vary based on experimental requirements, CCK-8 assays are an affordable and simple method of measuring cell viability in vitro.

Protocol for CCK-8:

Cells can be plated in a 96-well format and incubated at standard conditions (37˚C, 5% CO2). Typically, 1000 cells/well offer best results, but the number of cells may need to be optimized depending on the cell line used. Add 10 ul of CCK-8 reagent to cells in each well and incubate for 1-4 hours at standard conditions in an incubator. After an incubation period, measure absorbance at 450nm in a cell plate reader. To measure cytotoxicity, add various concentrations of desired reagent or drug into wells at the same time as CCK-8 reagent addition.

Advantages of CCK-8 Assay:

Cell Counting Kit-8 (CCK-8) offers numerous advantages compared to other cell viability or proliferation assays. CCK-8 assays do not use radioactive materials, are not toxic to cells in culture, and are extremely easy to use in the lab. CCK-8 is a one-reagent experiment that does not require any mixing, thawing, or preparation before use. Additionally, only 10μL is needed per well in a 96-well format. Once CCK-8 is added to cells, there are no washing or additional steps required to produce the colorimetric change. After a 1–4-hour incubation, plates are easily read in a cell plate reader at 450nm for results. Unlike many other assays, CCK-8 assays boast the ability for cells to be used in other experiments after the addition of the CCK-8 reagent due to the extremely low cell toxicity. For example, cells plated can first be used in a CCK-8 assay, and once results are obtained and media is changed, cells can conveniently be used in other assays. For your next cell viability or cytotoxicity assay, consider using Boster Bio’s Cell Counting Kit-8 (CCK-8) (Catalog# AR1160) for a simple and affordable assay.