| Question |
Answer |
| How many cells should be seeded per well? |
In a standard 96-well plate, seed at least 1,000 adherent cells per well in 100 µl of medium. Sensitivity is lower for leukocytes, so we recommend at least 2,500 cells per well in 100 µl. For 24- or 6-well plates, scale the seeding density accordingly and add CCK-8 at 10% of the total medium volume in each well. |
| Can I use a 384-well plate? |
Yes. Add CCK-8 at 10% of the medium volume. If that volume is too small to pipette accurately, dilute the CCK-8 solution 1:1 and add 20% of the medium volume instead. |
| Can I use a 24-well plate? |
Yes. Add CCK-8 at 10% of the medium volume in each well. |
| Does phenol red interfere with the assay? |
No. The absorbance contributed by phenol red is cancelled out when you subtract the blank well reading. |
| Do CCK-8 results correlate with thymidine incorporation assays? |
Yes, they correlate. Note, however, that the two assays measure different things, so the results may not be identical. |
| Can CCK-8 be used with bacteria? |
It works with E. coli but not with yeast. Add 10 µl of CCK-8 to 100 µl of E. coli culture and incubate for 1–4 hours, or overnight. |
| How stable is CCK-8? |
CCK-8 is stable for at least six months at 0–5 °C, and for one year at −20 °C protected from light. For long-term storage we recommend −20 °C. |
| What if I do not have a 450 nm filter? |
Any filter between 450 nm and 490 nm can be used. |
| How can I reduce variability caused by reagent residue on pipette tips or well walls? |
Dilute the CCK-8 reagent in medium and mix it before adding it to the wells. |
| What colour should CCK-8 be? |
It should be pink. A different colour may indicate the reagent has degraded, which can affect your results. |
| Does CCK-8 stain living cells? |
No. The active component is a water-soluble tetrazolium salt (WST-8), which is reduced by dehydrogenases in living cells via the electron carrier PMS. Because the resulting formazan is also highly water-soluble, it does not stain the cells. |
| Is the CCK-8 solution toxic to cells? |
The concentrated solution is slightly toxic because of its high PMS content, but once diluted tenfold in culture medium it is not. Long incubations — overnight or several days — are therefore possible, and the same culture can afterwards be used for other proliferation assays such as crystal violet, neutral red or DNA fluorescence. Tolerance varies between cell lines, so check viability after adding CCK-8 if you plan a long incubation. |
| Can test compounds interfere with the assay, and how do I deal with it? |
Sometimes. Reducing compounds react with CCK-8 and raise the absorbance. To check, add CCK-8 to medium containing the compound but no cells and read at 450 nm; if the absorbance is higher than for compound-free medium, the compound interferes. Replace the medium before adding CCK-8 to remove the effect. |
| Why do my readings vary between runs? |
Three common causes: (1) evaporation from the outermost wells changes the volume during incubation — fill the outer ring with medium only and do not use it for measurements; (2) reagent adhering to the well wall — tap the plate gently after adding CCK-8 to mix; (3) too many or too few cells per well — optimise within the range of 1,000–100,000 cells per well. |
| How do I set up the blank control? |
Add CCK-8 to cell-free medium, incubate for the same length of time and read at 450 nm. For cytotoxicity assays, also account for the test compound: add CCK-8 to cell-free medium containing the compound and use that as the blank. |
| What substances interfere with the assay? |
Reducing agents interfere — for example glucose solutions containing vitamin C. (Normal culture media contain too little to matter; phenol red and serum do not interfere.) Phenol red raises the blank absorbance but does not affect the result once the blank is subtracted. |
| The absorbance is too high but I cannot reduce the cell number. What can I do? |
Shorten the incubation after adding CCK-8 — for example from two hours to one. |
| What is the reference wavelength for, and is it required? |
It is not required. CCK-8 has no absorbance at the reference wavelength; the reference reading is used to subtract absorbance caused by turbidity in the sample. |
| The protocol covers 96-well plates. How much CCK-8 should I add in 24- or 12-well plates? |
As a general rule, add CCK-8 at one tenth of the medium volume. |
| How do I stop the colour reaction? |
For a 96-well plate, use any of the following: (1) move the plate to a 4 °C fridge; (2) add 10 µl of 0.1 M HCl per well; (3) add 10 µl of 1% (w/v) SDS per well. Read the plate within 24 hours of stopping the reaction. |
| Is pre-incubation of the cells necessary? |
Not strictly. Pre-incubation is recommended so that cells are in good condition; without it, intracellular dehydrogenase activity may be less stable. Some users skip it, but the standard curve and the assay must then be run under identical conditions. |
| Do metal-containing compounds interfere? |
Yes. At a final concentration of 1 mM, lead(II) chloride, iron chloride and copper sulphate inhibit colour development by roughly 5%, 15% and 90% respectively, reducing sensitivity. At 10 mM, inhibition is complete. |
| How should CCK-8 be stored? |
Protected from light, CCK-8 is stable for one year at 4 °C. For longer storage, −20 °C is recommended. Repeated freeze–thaw cycles raise the blank absorbance and affect results, so if you use the reagent regularly, keep it at 4 °C. |
| Do I need to count the cells when changing medium after pre-incubation? |
Normally it is enough to trypsinise cells in log phase, count them with a haemocytometer and prepare a suspension at the required density. If you need an accurate count, count the cells with a haemocytometer after pre-incubation. |
| Is the sensitivity the same for all cell types? |
No. Suspension cells are harder to assay than adherent cells. Adherent cells usually give a strong absorbance after 1–4 hours; suspension cells may read lower, which can be improved by extending the incubation or increasing the cell number. |
| Do suspension and adherent cells need different cell numbers? |
Suspension cells are harder to assay and generally need a higher cell number and a longer incubation. Adherent cells react readily; with too many cells the absorbance can exceed the plate reader's range. |
| Should I run a standard curve every time? |
We recommend it. Even with the same cell line, cell condition varies between experiments. Sensitivity can also differ slightly between reagent lots, so run a separate standard curve for each lot. |
| If the cells are dead but dehydrogenase activity remains, can I still calculate cell number? |
No. CCK-8 measures viable cells indirectly through dehydrogenase activity. If the enzyme is still active in dead cells, the assay will overestimate the number of viable cells; use a different method in that case. |
| Should I check whether my medium reacts with CCK-8 before starting? |
Yes. Test one well first, because some media contain redox-active substances. A normal reading should be below OD 0.4. |
| What if the OD is too low? |
Two options: increase the cell number, or extend the incubation after adding CCK-8. |
