Sensitivity
CCK-8 (WST-8) is the highest sensitive dye for the cell based assay.



Fig. 3 Comparison of the sensitivity using tetrazolium dye

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Cytotoxicity of Reagents
Only in CCK-8, continuous culture is possible without killing cells.

Fig. 4 Observation of Cytotoxicity after incubation with each reagent.
HeLa cells were incubated with WST-8 (CCK-8), WST-1, or MTS at 24 hours from addition of those reagents.

Example Data using Cell Counting Kit-8


Fig. 5 Data for Cell Proliferation and Cytotoxicity Assay

References

How many cells should there be in a well?
For adhesive cells, at least 1,000 cells are necessary per well (100 μl medium) when using a standard 96-well plate.
For leukocytes, at least 2,500 cells are necessary per well (100 μl medium) because of low sensitivity.
The recommended maximum number of cells per well for the 96-well plate is 25,000. If 24-well or 6-well plates are used for this assay, please calculate the number of cells per well accordingly, and adjust the volume of the CCK-8 solution in each well to 10% of the total volume.

Can this kit be used with a 384-well plate?
Yes, you can use this kit for a 384-well plate. Add CCK-8 solution with 1/10 volume of the culture medium in a well. If the volume of CCK-8 to be added is too small, dilute CCK-8 in half with the medium and add 1/5 of the volume of culture medium in a well.

Can this kit be used with a 24-well plate?
Yes. Add CCK-8 solution with 1/10 volume of the culture medium in a well.

Does CCK-8 stain viable cells?
No, it does not stain viable cells because a water-soluble tetrazolium salt (WST-8) is used for the CCK-8 solution. The electron mediator, 1-Methoxy PMS, receives electrons from viable cells and transfers the electrons to WST-8 in the culture medium. Since its formazan dye is also highly water soluble, CCK-8 cannot be utilized for cell staining. 

Does phenol red affect the assay?
No. The absorption value of phenol red in culture medium can be removed by subtracting the absorption value of a blank solution from the absorption value of each well. Therefore, culture medium containing phenol red can be used for the CCK-8 assay.

Is there a correlation between CCK-8 and the Thymidine incorporation assay?
Yes. However, please note that since CCK-8 uses a different assay mechanism from that of the Thymidine assay, so the results may differ. Comparison data are shown in the technical manual, which is available at www.dojindo.com/tm.

Is CCK-8 toxic to cells?
CCK-8 solution itself is cytotoxic because of the high concentration of 1-Methoxy PMS. CCK-8 in the culture medium, however, is not cytotoxic because of the ten times dilution with culture medium. Therefore, long incubation, such as overnight and several days, is possible. The same cell culture can be used for other cell proliferation assays, such as the crystal violet assay, neutral red assay, or DNA fluorometric assays after the CCK-8 assay is completed. Since each cell has a different tolerance to CCK-8, incubate cells with CCK-8 solution and check the cell viability if a longer incubation time is necessary.

How stable is CCK-8?
CCK-8 is stable over 6 months at room temperature and one year at 0-5 oC with protection from light. For longer storage, we recommend storing the solution at -20 oC ; CCK-8 is stable over 2 years at -20 ^oC.

I do not have a 450 nm filter. What other filters can I use?
You can use filters with an absorbance between 450 nm and 490 nm.