Background: Determining the cell # is based on various cell functions, such as enzyme activity, cell membrane permeability, cell adherence, ATP production, co-enzyme production, and nucleotide uptake activity. A variety of methods such as Colony Formation, Crystal Violet, Tritium-Labeled Thymidine Uptake, MTT, and WST, had been developed for counting live cells. 

Trypan Blue: can not distinguish healthy cells and the cells that are alive but losing cell functions. 

Tritium-Labeled Thymidine Uptake: sensitive to determine the influence on the DNA polymerization activity, but it requires radioisotope which causes various concerns. 

Cellular enzymes: lactate dehydrogenase (LDH, not stable when cells are dead), adenylate kinase (not stable when cells are dead), and G6PD are used as cell death markers. 

Enzyme-based: MTT & WST. MTT is for determining the mitochondrial dehydrogenase activities in the living cells. MTT is reduced to a purple formazan by NADH. However, MTT formazan is insoluble in water, and it forms purple needle-shaped crystals in the cells. Therefore prior to measuring the absorbance, an organic solvent is required to solubilize the crystals. Additionally, the cytotoxicity of MTT formazan makes it difficult to remove cell culture media from the plate wells due to floating cells with MTT formazan needles, giving significant well-to-well error. 

Cell Counting Kit-8 (CCK8): use a tetrazolium salt (WST-8), which produces the water-soluble WST-8 formazan (orange color, received two electrons from viable cells through electron mediator, 1-Methoxy PMS by NADH and NADPH activity). The amount of WST-8 formazen is dependent on the activity of cellular dehydrogenase, so WST-8/1-Methoxy PMS system can be used to determine the # of living cells and cell viability.  WST, WST-8 formazan, and 1-Methoxy PMS are highly stable, and non-toxic to the cells. 

WST-8 is not cell-permeable, so it will result in low cytotoxicity. 

CCK-8 is widely used for Cell Proliferation Exp, Cytotoxicity test, drug sensitivity test. 

It's necessary to have a proportional relationship between absorption and viable cell numbers. It is desirable to start with a set number of cells, and then determine the suitable incubation time for color development. Even when the cell number is the same, HeLa cells and HL60 cells have quite different cell activities (which means the absorbance is very different). So, in a preliminary exp, it is recommended to determine the suitable conc. of cells for each cell type and the time of coloration.  

Q: what is the toxicity of CCK-8 compared to MTT?

A: Compared to MTT (MTT can enter the cells) in which the cell can not survive after the reagent had been added, the cell survial rate for CCK8 is >90% after 25h incubation. Therefore, after the assay with CCK8, those cells can be used for another exp. However, it is necessary to wash the cells so that no dye remains on the cell surface. 

Q: When using CCK8, what # of cells is appropriate? 

A： The appropriate # of cells depends on the type of cells and the type of your exp. The amount of coloration will differ depending on the cell type, even if the cell # per well and incubation times are the same. When using a 96 well microplate, please check the absorbance level of 1,000-25,000 cells/well. If the experiment is for a toxicity test, 5,000-10,000 cells/well may be appropriate. If the # of cells is expected to increase during the assay, prepare a plate with 1,000-5,000 cells/well.  

Q: How long of a pre-incubation time is required prior to the assay?

A: It depends on the cell type, but the cells should enter into the logarithmic growth phase. The average incubation time to enter into this phase is 24h-48h.

It is recommended to pre-incubate the adherent cells. When collecting the cells from a culture flask using Trypsin, the activity of the cells is not normal. Because of this, it is necessary to pre-incubate to get the cells back to their logarithmic growing phase to regain viability prior to the assay. For non-adherent cells, you can skip this step if the same culture medium is used for harvesting and resuspending cells for the assay. 

Q: How much incubation time is sufficient for color development? 

A: In general, the incubation time is 1-4 hrs. However, the absorbance will differ between cells types even if the # of cells/well and coloration time are the same. Set an appropriate incubation time to give a proportional relationship between the cell # and the absorbance. 

Q: The cell culture is not clear, and has some turbidity. 

A: measure the absorbance at 600-650nm of the well as a reference. Then, the absorbance at 600-650nm should be subtracted from the absorbance of the same well measured at 450nm to eliminate the background that comes from turbidity.