Hi all,
Can I use the same cells again after I put them in 96-well plates, tested them with a cell counting kit at OD450 (CCK8) at 24 hours, washed them with PBS, added new media, and let them sit for another 24 hours to get a 48-hour time point, and did the same thing for another 24 hours to get a 72-hour time point, or do I need to do three parallel plates to get three timepoints?
Hello Sya Retno
You need not do three parallel plates to get three timepoints.
Cell Counting Kit-8 (CCK-8) uses a tetrazolium salt, WST-8, which produces the water soluble WST-8 formazan. Since this orange colored formazan does not require dissolving, no solubilizing process is required and therefore no extra solubilizing solution need to be added to the wells. Moreover, WST-8 is not cell permeable, which results in low cytotoxicity. In CCK-8, 1-Methoxy PMS receives an electron from NADH or NADPH at the membrane or inside of the cell and passes the electron to the WST-8 that is around the outer cell membrane. WST-8, WST-8 formazan, and 1-Methoxy PMS have no cytotoxicity in cell culture media.
Therefore, after the assay, it is possible to continue further experiments using the same cells. However, you should wash the cells with PBS (which you have already done) so that no dye remains on the cell surface.
Best.
Hi Sya,
This has been a question for me for a while because I want to save some materials I prepared.
CCK-8 can cause significant intracellular metabolic changes, particularly the down-regulation of metabolites within the glycolysis and pentose phosphate pathways. I strongly advise exercising caution when using cells treated with CCK-8 in subsequent experiments, as these metabolic changes could impact experimental outcomes and interpretations. This also depends on the drug/materials/treatments.
I would recommend NOT continuing further experiments using the same cells if you are not sure about the changes that happen at the molecular level by the treatment you applied.
Best.
REF:Article Cell via Cell Viability Assay Changes Cellular Metabolic Cha...
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