Cell counting Kit-8
Cell counting Kit-8 is also known as CCK-8, which is used to determine the viability of the cell on the basis of the calorimetric assay. Thus, it can also be used in assays related to cellular proliferation and cytotoxicity. These cellular toxicity and viability assays determine the viability or non-viability of the cell in the given sample by the range of mechanisms. It is also an indicator of cellular proliferation. These cytotoxicity assays determine the change in the structural integrity of the cell, like loss of membrane permeability or integrity, the ability of the cell to reduce any substrate, and several enzymatic processes. Upon cellular death, there will be a loss in membrane integrity that results in the out flux of cellular parts and enzymes along with the influx of substrates. Cell counting Kit-8 is based on this principle. This kit requires the use of tetrazolium salt. This salt is water soluble in nature, so upon contact with the cell, due to the dehydrogenase activity of the cell, the cell converts this tetrazolium salt to formazan dye which gives yellow colour. All this happens due to the redox activity by the cell. Thus, it can be used to determine the number of live cells. The increase in the dye is directly proportional to the viability of the cell. It means as the number of live cells is high, then there will be an increase in the dehydrogenase activity of the cell, that in return convert more tetrazolium salt to yellow colour dye (formazan). Hence more yellow color or dye formation indicates a greater number of live cells within a sample.
Similarly, several Studies introduce the use of cell counting Kit-8 for the detection and measurements of live bacteria within the samples. As we know, pathogenic bacteria are low in number but their resistance is High. So, there must be the detection of the bacteria more accurately. In the past, several methods include, polymerase chain reaction (PCR), turbidimetric method, antibody-based method, plate count methods, and nucleic acid amplification were in use. But all of them have some drawbacks. The most commonly used technique turbidimetric method is simple to use but it can not differentiate between live and dead bacteria. On the other hand, the plate counting method is considered a gold standard test for viable bacterial detection and gives colony-forming units (CFU). But the problem is that it requires many sample dilutions along with a greater culturing time of almost 1–2 days. Similarly, nucleic acid amplification is also a laborious method to do. It is because it requires several steps to perform. Also, before treatment, this method requires pretreatment steps to perform. These steps include the extraction of DNA prior to the performance along with the amplification of signals by the process of the thermocycler. Therefore, there is a need to develop a method that is most reliable in use and can count a viable number of bacteria by either evaluating electron transport or reducing ability. It can be done through calorimeter assays which require 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). But the problem is that this reagent, upon reaction, produces low water-soluble formazan, and also tetrazolium is somewhat toxic. So, cell counting Kit-8 which is based on WST-8 and electron carrier (1-methoxy PMS), can be used for detection.
Figure 1:Diagrammatic representation of the detection of live bacteria via dehydrogenase activity using cell counting Kit-8. (CCK)
In this study, Researchers used staphylococcus aureus and E. coli bacteria as model organisms to find out the optimal conditions for the assay to determine the viability and to determine the feasibility of this assay. This method gives the maximum absorbance of 450nm (OD450nm) in about 2 hr. So, 2 hr is sufficient to detect these viable bacteria in the sample. This absorbance value (OD450nm) is correlated with the number of live bacteria in a given sample. And the value of OD 450nm is dependent upon the amount of formazan which is formed by the reduction reaction carried by dehydrogenase enzyme with tetrazolium. So, studies suggest the use of CCK-8 for the detection of viable bacteria because of giving satisfactory results with high sensitivity. And the results are explained based on absorbance value. As the number of live bacteria in the sample increase, there will be increased production of formazan, that in turn give the increased value of absorbance(Xianhong Yang, 2021).
As we have already discussed that this kit can be used to measure cellular proliferation. Studies reported that the CCK-8 assay along with the use of drug lymphocytes stimulation test can be used for the diagnosis of liver injuries that are induced by the drugs(ZuoPeng et al., 2016).
Similarly, various studies have been conducted in which researchers used cell counting Kit-8 assay to detect the inhibitory effects of drugs in various diseases or to determine the evaluation of the anti-cancer drug. Researchers have conducted various trials in which they evaluate the 5-Aza-2’-deoxycytidine which has inhibitory effects on chronic myeloid leukemia cells. They use CCK-8 to detect the proliferation of K-562 cells. They found that when the k562 cells come in contact with the 50% 5-Aza-2’-deoxycytidine with an inhibitory dose of 15.55nmol/L, there will be the arrest of the cell cycle at the G2 phase, which leads to a decrease in proliferation and finally the cells undergo apoptosis. Hence we can use the cell counting Kit-8 assay to determine the effects of this drug on k-562 cells(MA., 2014).
The cell counting Kit-8 has various advantages to use in cell toxicity and proliferation to determine the viability of the cells. Methylene blue is considered toxic to nucleus pulposus cells. Studies suggest that cellular toxicity can be measured via cell counting Kit-8 assay. In study researchers divided these samples into 9 groups for the purpose of culture. These groups include blank control that has only cells and medium, control that has cells, medium, and CCK-8, and the methylene blue groups with different concentrations. They observe that the control group shows the darkest color, while the methylene blue group shows the lighter color as its concentration increases. It is due to the cellular viability, which means the cells which have a high level of methylene blue become dead, that in turn leads to less absorbance value than that of the sample which has less level of methylene blue. This is how the cell counting Kit-8 is helpful in measuring the viability of cells(Hua-long, 2018).
References
HUA-LONG, F. 2018. Quantitative study on cytotoxicity of methylene blue to human nucleus pulposus cells by cell counting-kit 8 assay.
MA., Y. L. C. S. J. L. H. Y. Y. S. W. 2014. Application of Cell Counting Kit-8 in detecting the growth inhibiting effect of 5-Aza-2 ’-deoxycytidine on chronic myeloid leukemia cells
XIANHONG YANG 2021. A simple colorimetric method for viable bacteria detection based on cell counting Kit-8.
ZUOPENG, F., SHAN, L., WEI, N., LIXIA, C., JIE, C., JING, Z. & RONGHUA, J. 2016. Application of drug lymphocyte stimulation test using cell counting kit-8 assay in diagnosis of acute drug-induced liver injury. 临床肝胆病杂志, 32, 1562–1565.
