I am currently using the MTT assay to study the effect of a selected drug on the cell proliferation rate of carcinoma cell lines. I encountered a problem here, which is cells treated with drug show similar results at different concentrations of drug. the results of cell viability at different concentrations are about 40%.
Cell viability (%)
25µM 40.49%
50 µM 35.2%
100µM 34%
150 µM 35%
Can any experts help?
Hi Somayyeh,
First of all MTT, is not so reliable assay. Still it is first line of experiment which most of the researcher start their anti-cancer related work. It measures the ability of cells to reduce the tetrazolium dye and gives the metabolic activity of live cells.
But, it has some key limitations such as,
1. Assay won't works with complete cell growth pattern, its measuring activity at the most during growth phase.
So, your cells number and doubling time should be optimized before. Otherwise, you cannot chase the changes caused due to your drugs
2. It is determined using absorbance, so is not very sensitive in term of activity you are looking since it won't track the minor changes. Assay doesn't fit completely with proliferation, because maybe due your drug treatment cells died, but not completely, the unaffected cells may strongly convert tetrazolium dye to formazan, and final output between control and 150uM drug treated cells coming like similar no change.
Try for others cyotoxicity assays. PFA
https://www.ncbi.nlm.nih.gov/books/NBK144065/
what is the diluent for drug. if it is DMSO it should be less than 0.1%.check your DMSO concentartions
Dinesh Kumar Parandhaman
yes, it is DMSO and its concentration is less than 0.1%.
It is not possible to give you a reasonable answer on the basis of the provided information. If you're expecting changes in cell proliferation, you should count your cells. The MTT-assay is a metabolic assay and may be affected by the drug directly.
This issue has been discussed on Research Gate before - perhaps previous comments could also give you some help to understand your problem.
https://www.researchgate.net/search.Search.html?type=question&query=MTT%20assay
did you cross check with trypan blue stain. At times i have compared both MTT and trypan blue assay outcomes. if by trypan blue also it is same. then
1. how old are your cells( how many passages it has been done) more passages lead to problems in assay.
i have encountered this with erk signaling pathways.
2. try using different kit from another company . but be sure that your cells are healthy
It may be possible to obtain similar results because of the low solubility of this drug in DMSO?
How long you are keeping after DMSO addition for purple color development (means dissolving of formazan)???
Sorry Somayyeh Mirzaeinia I am wiillig to know the time duration, you are keeping the sample in DMSO for purple color development(Formazan dissolving). I got your point that you keeps the samples with MTT for 4hr in dark (means in incubator). after that we adds DMSO. So, after addition of DMSO and how long its taking to develop color or how long you are keeping in DMSO for color development and goes for reading measurement. That particular time duration.
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