I'm analysing whether a new compound is able to make HCT116 cells more sensitive to X-ray radiation and tested different concentrations in combination with different doses of irradiation. However, when analysing the viability using an MTS assay, I don't see any irradiation dependency. It looks as if the cells only react to the compound but not to the X-ray at all, the viability is the same for 0, 2, 4, 8 or 10 Gy. Did anyone experience similar results or does anyone have a suggestion what I could have done wrong?
Thank you!
I'm using MTS assays from Promega and I can garantee it power and sensitivity but I'm working with chemical compounds. Have you checked for bacterial contamination in the medium? because bacteria are able to metabolize the tetrazolium too generating the salt and so you see no differences.
Have you checked with a microscope if treated cells are less then untreated one? maybe your treatment is not cytotoxic at the doses you tested
Generally, short-term MTS assays underestimate cell killing by radiation. It would be best to use the gold-standard clonogenic survival assay when looking at radiosensitization. You can read more about this in the attached paper.
Article Brown JM, Wouters BGApoptosis, p53, and tumor cell sensitivi...
Thank you very much for your answers!
@Diego: I've checked for bacteria but it seems all clean. Yes, I use Promega as well and for chemical compounds its really good. Probably Sandro and Ronald are right, I will just have to do a long-term clonogenic assay to really assess the killing by radiation - didn't think that the short time span of MTS assays might be the problem. Thanks again :)
Well MTT is established as somehow proliferation assay, but if you go in detail to the mechanism, it just reflects the situation of your reducing equivalents. Probably, your cells show a decrease in metabolic activity which reflects a non-difference in your OD value after performing MTT incubation. Did you already checked your cells for apoptosis after IR? Probabaly, you can differentiate between early and late apoptosis after IR and forget proliferation. Otherwise you can try BrdU.
Thanks for your answer Christian! Yes, I checked for apoptosis and see an increase using Annexin V/PI and flow cytometry. That's why I was surprised about my MTS assay outcome. But at the end, I kind of understand your comment similarly to the other ones, MTS (or MTT) might not be sensitive enough since it is too short term. I am doing a clonogenic assay at the moment and hope this will give me a better answer.
Perhaps you also can try an assay for detection of mitochondrial membrane potential as an early step of intrinsic apopotsis after IR. For this TMRE is sufficient and easyily to handle. It also reflects metabolic drifts after IR.
Nice suggestion, never came across this kind of assay. Thanks Christian!
I also use Promega MTS and have looked at both chemo and radio treatments. However as others have suggested alongside this I have also done clonogenic assays.
Specifically with the MTS I did see a reduction in responses at the 2Gy, 10Gy and 5x2Gy schedules. However this was also cell line dependent as some were more resistant than others.
Drop me a message if you wish to discuss further.
Different methods look at different modes of cell death or inactivation, and several experimental approaches may be chosen depending on the purpose of studies. For instance, MTS, MTT, Alamar Blue etc evaluate reflects changes in cellular metabolic activity, whilst clonogenic survival assay evaluate changes in cellular clonogenic potential. Senescent cells are metabolically active, but clonogenically inactive.
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