I have performed a MTT viability assay to see how my cells (MCF-7 and HCT-116) respond to a drug. I had one control with cells + medium and another control with cells + medium + DMSO in different concentrations, the cells treated with the compound (wich is dissolved in DMSO) showed more viability than the cells treated with DMSO only in the same concentration. How is that possible? Is that normal to happen in a MTT assay?
Thanks!
How do the control (cell plus medium only) compare with the DMSO control (cells plus medium containing DMSO)? Is there a difference. My previous experiments with MCF7 showed no difference between the two controls if DMSO was below 0.1 %. Your compound may actually stimulate mitosis thus giving you higher readings than the control.
Did you dissolve your DMSO in medium prior to adding to your cells? Also, is the final concentration less than 1%?
Hello, Jordan. Thanks for answering.
Yes, I did dissolve the DMSO in medium. I used the following final DMSO concentrations: 0.004%, 0.01%, 0.02%, 0.04%, 0.06% and 0.08%
Is it true in all cases that the DMSO is more toxic? Maybe you could try bringing DMSO up to 0.08% in all drug conditions.
How do the control (cell plus medium only) compare with the DMSO control (cells plus medium containing DMSO)? Is there a difference. My previous experiments with MCF7 showed no difference between the two controls if DMSO was below 0.1 %. Your compound may actually stimulate mitosis thus giving you higher readings than the control.
Hello, Edmund, thanks for your answer. I am getting around a 90% viability comparing my vehicle control (DMSO + medium + cells) with my control (Cells + medium). I think my compound could be stimulating mitosis in MCF-7 but not in HCT-116. Is that possible that the compound acts differently between the cell lines?
It is very possible. I did some experiments with estrogen receptor positive (MCF7) and receptor negative cells. MCF7 cells with DMSO +estrogen gave higher MTT readings than control, while the receptor negative cells were the same as control.
Gabriel,
Did you do cytotoxicity assays manually (I mean using trypan blue and eg. Burker haemocytometer)? As you know, MTT assay reflect cells' metabolic activity rather than cell count. So, theoretically, you can imagine situation that less in number but more metabolic active cells reduce more MTT than more in number but much less in activity ones, can't you? Try to do it as I mentioned above - you will see the morphology of your cells (which give you some important informations about the cells) plus you will get the answer about cytotoxicity of your compound(s) regarding the number of cells.
Just a thought, but have you tried EtOH instead of DMSO as your solvent carrier? I have worked with RAW 264.7 cells, which are notorious for sensitivity to DMSO, even at micromolar concentrations (causing them to differentiate into osteoclasts). The basis for this is not known, but DMSO may potentiate M-CSF and/or RANKL effects. This is just to say you can't necessarily regard DMSO as a benign carrier (even apart from toxicity at higher concentrations), and that it's effect can be quite cell specific.
Robert, thanks for your advice. I will count them using trypan blue, I haven't done it yet because I am a little skeptical regarding manually analysing cell viability, but that could be a great way to see if it matches my MTT results.
Norbet, I haven't tried EtOH instead of DMSO because the Lab who produced the compound told us to use DMSO as the solvent, but I guess I could make an experiment using the EtOH to see if affects my results. Thank you!
Gabriel, if it is a precious or expensive compound, do a solubility test first with a small amount. Some compounds that dissolve in DMSO are not very soluble in EtOH. Good luck!
Gabirel, give it a try. But remember that MMT and trypan blue results may (and most probably will) differ. These are different assays based on different principles. All depends on the compound you work with. I agree that counting cells manually is very difficult (and boring) as you must be full focused all the time as well as the intra-assay error is high (up to 20% because of the "human factor") - every single cell missed or double counted in Burker haemocytometer changes your result by 2500 cells/uL. But despite all that, sometimes this procedure will help to find some answers, like in your situation. Good luck!
In addition to valuable advice given above, I would also like to point out that Inhibition of efflux proteins is associated with increased reduction of MTT, implying an apparent increase in cell viability. See our publication Vellonen et al. (2004) Eur J Pharm Sci 23:181 and http://www.ncbi.nlm.nih.gov/pubmed/15451006
Best regards,
Paavo
I agree with Robert, the MTT assay may not be the best for your experiments. I ran into similar problems... I based my hypothesis on the results I had from such an experiment and when I was asked to confirm with viability dye and a separate proliferation dye using flow cytometry the results were different. I realized the plate based assay had some limitations that were not conducive for my cells (splenocytes) - the difference was clearer after my flow cytometry analysis.
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