Hi,
Currently I am working with a compound. The compound at 1 uM exhibits no toxicity using CCK8, however according to Cell Titer Glo, the compound at 1 uM kills 80% of cells. Why do I get different results? Has anyone had a similar problem? CCK8 is based on reduction of NAD of NADH. Is it possible that this compound affects this process, and kills cells simultaneously?
Thanks
Burak
Well, as you say: each kit depends upon different enzymes. CCK8 depends upon dehydrogenase enzymes within the cell, while Celltiter glo depends upon luciferase you add with the reagent mix. You could be inhibiting the dehydrogenases for CCK8 in the first case, or you might be competing with the ATP needed for luciferase in celltiter glo. The latter would be comparatively easy to test - just run an assay with your compound, Celltiter glo, and ATP, and see if the reaction is inhibited or not. The former is a bit harder to test directly, unfortunately.
You could also have a more fundamental problem - all these kits claim to measure cell viability, but what they're really measuring is some specific chemical process. And while these processes TEND to correlate highly with cell number, once you start adding inhibitors to the mix, you might inhibit one and not the other. In this case, you might be messing with ATP generation while not significantly effecting NAD/NADH balance.
Finally: it could just be that one reagent works better for your particular cell line than the other. Have you done a simple cell density curve, to ensure that you get linear readings across the range of cell numbers you're measuring? If you're not inside the linear range of the assay, that might explain your odd results.
If everything else fails, I would try complementing the assay by simply counting the cells in each condition - all these reagents are essentially trying to be as accurate as cell counting, after all. And while it's tedious, it's also comparatively unambiguous.
As Mr. Katt already explained very well, the two kits are measuring different things. We prefer not to use the term "viability", but "metabolic activity" when refering to the GloAssay.
Cell counts, best with Trypan Blue exclusion, are indeed very accurate if you want to determine cellular viability.
It is very well known that all these different viability assays might lead to different results, depending on the drug or compund you want to test and its mechanism of action.
Based on my experience and extensive research, I have encountered a similar issue and would like to provide an explanation for the following question, even though my response may be late.
The discrepancy in the results obtained from CCK8 and Cell Titer Glo assays could be due to various factors. It's important to consider the specific mechanisms and limitations of each assay to understand the differences observed.
- CCK8 assay measures the reduction of a tetrazolium salt (WST-8) to formazan by cellular enzymes, including NAD(P)H-dependent dehydrogenases. The amount of formazan produced is directly proportional to the number of metabolically active cells.
- Cell Titer Glo assay measures the amount of ATP (adenosine triphosphate), which is an indicator of overall cell viability. The assay uses a luciferase-based reaction to quantify ATP levels.
- CCK8 assay is widely used for assessing cell viability, but it is primarily based on the metabolic activity of cells. It may not detect certain forms of cell death that do not involve metabolic inhibition.
- Cell Titer Glo assay, on the other hand, directly measures ATP levels and provides a more comprehensive assessment of cell viability. It can detect cell death caused by various mechanisms.
- Since CCK8 assay relies on the reduction of NAD(P)H, it is possible that your compound affects this metabolic process or the activity of dehydrogenases involved. This interference could lead to an underestimation of cell death using the CCK8 assay.
- The Cell Titer Glo assay measures ATP levels, which are essential for cell survival. If your compound affects ATP production or stability, it could result in a higher detection of cell death using this assay.
To address this issue and gain a better understanding of the compound's effects, you can consider additional experiments or assays to assess cell viability and death. Examples include trypan blue exclusion assay, propidium iodide staining, annexin V staining, or caspase activity assays, depending on the specific characteristics of cell death you want to investigate.
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