Storage Condition : 0-5oC
Shipping Condition : ambient temperature
Product Description
Cell Counting Kit-8 (CCK-8) allows sensitive colorimetric assays for the determination of cell viability in cell proliferation and cytotoxicity assays. Dojindo’s highly water-soluble tetrazolium salt, WST-8, is reduced by dehydrogenase activities in cells to give an orange-color formazan dye, which is soluble in the tissue culture media. The amount of the formazan dye, generated by the activities of dehydrogenases in cells, is directly proportional to the number of living cells. The detection sensitivity of CCK-8 is higher than the other tetrazolium salts such as MTT, XTT, MTS or WST-1.
Cytotoxicity Test for 3D Cultured Model
The cytotoxicity test method for 3D cultured model using Cell Counting Kit-8 has been added to OECD TG492 as the standard method. For details, please see the following page.
<OECD website>
Cell viability detection mechanism with CCK-8
Procedure
Cell Counting Kit-8 requires 3 simple steps.
Sensitivity
CCK-8 (WST-8) is the highest sensitive dye for the cell based assay.
Comparison of the sensitivity using tetrazolium dye
Cytotoxicity of Reagents
Only in CCK-8, continuous culture is possible without killing cells.
As seen in the photos, even after 24 hours of incubation with CCK-8,
cells continue to proliferate. Other cell viability assay kits are toxic to
cells, and most of the cells are killed during the first 3 hours of incubation.
Stability of Reagent
With stability up to a
year at 5oC, you do not have to worry about preparing your cell
proliferation reagents right before your experiment. Cell Counting Kit-8 is
even stable for 6 months at room temperature. There is no one out there who can
survive the heat like us.
Example Data using Cell Counting Kit-8
Absorption spectrum
Absorption spectrum of WST-8 formazan dye
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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?
Since the toxicity of CCK-8 is so low, the same cells can be used for other cell proliferation assays such as the crystal violet assay, neutral red assay or DNA fluorometric assay after the CCK-8 assay is completed.
How stable is CCK-8?
CCK-8 is stable for one year at 0-5oC with protection from light. For longer storage, we recommend storing the solution at -20oC.
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.