Cell Counting Kit-8 FAQs

1. How many cells should there be in a well?

For adhesive cells, at least 1000 cells are required per well (100 μl medium) when using the kit's standard 96-well plate. For leukocytes, at least 2500 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 25000. If a 24-well or 6-well plate is used for this assay, please calculate the number of cells per well accordingly, and adjust the volume of the CCK-8 solution in a well to 10% of the total volume.

2. Does CCK-8 stain viable cells?

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

3. How stable is CCK-8?

CCK-8 is stable for 2 years at -20 °C, 1 year at 4 °C, and 3 months at room temperature. CCK-8 is stable over 2 days even at 60 °C as long as the CCK-8 solution keeps its original red color and does not turn orange.

4. Does phenol red affect the assay?

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

5. Is CCK-8 toxic to cells?

The toxicity of CCK-8 is so low that, after performing the assay, the same cells can be used for other cell proliferation assays such as the crystal violet assay, neutral red assay or DNA fluorometric assay.

6. I do not have a 450 nm filter. What other filters can I use?

Although 450 nm filter gives the best sensitivity, you can use filters with the absorbance between 450 and 490 nm.

7. Can I use CCK-8 for 384-well plates?

CCK-8 can be used for 384-well plates. Please dilute the CCK-8 solution with PBS; the required volume of the CCK-8 solution is 5 μl per well for the 384-well plate.

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