while doing my experiments on VERO cells, testing a radioprotective agent 24 h after irradiation (6 Gy), i run an MTT assay and i found viability above 100% (around 138%) and i couldn't explain it.
any idea
Hey ,
MTT test is dependent on mitochondria activity ... maybe you increased the mitochchondria growing with the radioprotective agent ;)
best regards
Two questions Herrrati,
Which was the radioprotective agent you applied to the cells? Perhaps it could be inducing a proliferative effect.
What was the viability percentage obtained for the non-treated cells?
It could be good to repeat the same MTT assay but also with cells treated with some agent known proliferative effect, as positive control.
María Luisa Caballero thank you so much for replying, well the radioprotective agent is an extract of a plant (mainly flavonoides and polyphenols) and the percentage obtained for the non-treated cells was around 88.06% for the 6Gy irradiation
It seems clear that a mitochondrial activity increased with this extract applied to the cells.
A quick literature revision about flavonoides and polyphenols used in MTT assays show the following as expressed in the discussion of a the paper [Bernhard D, Schwaiger W, Crazzolara R, Tinhofer I, Kofler R, Csordas A. Enhanced MTT-reducing activity under growth inhibition by resveratrol in CEM-C7H2 lymphocytic leukemia cells. Cancer Lett. 2003;195:193-9], "based on the currently available information, a possible pitfall of the MTT assay seems to be restricted to polyphenols with antioxidant properties. At a lower, pre-apoptotic, possibly differentiation-inducing concentration range, these compounds – in spite of growth inhibition – can induce in certain cell types an increase of the MTT reducing activity which is not related to the number of living cells. The result of this is the presence of an increased MTT-reducing activity in a slower growing cell fraction, compared to the faster growing untreated control cells".
In this sense, It is also interesting the article: Wang P, Henning SM, Heber D. Limitations of MTT and MTS-based assays for measurement of antiproliferative activity of green tea polyphenols. PLoS One. 2010 Apr 16;5(4):e10202.
To verify this possible effect of the extract you used and to be sure the experiment is working well, it would be necessary repeat the MTT assay with other agents as positive and negative control.
Yes, it is a very useful cell viability assay manual. Moreover, as Dr Ganguly suggests, It would be a good idea perform combined Crystal Violet/Neutral Red/MTT assays [Chiba K, et al. Simultaneous evaluation of cell viability by neutral red, MTT and crystal violet staining assays of the same cells. Toxicol In Vitro. 1998;12:251-8].
This depends on your control and reliability of the experimental setting and method (including controls)
MTT is a less sensitive method compared to other available assays for analyzing cell viability still obtained cell viability is well documented but do not expect the answer about the proliferation of cells directly from MTT. It is cell number which is determining factor in MTT. The healthy and rapidly growing cells exhibit high rates of MTT reduction to formazan while the dead or inactive cells fail to do so.
If you are using MTT assay and observing the difference in viability then majorly its due to the change in mitochondria activity, not due to the change in amount of cells. So you need to think very carefully what your assay measures. All around, keep additional controls to get result without any doubt.
Honestly, try some other sensitive assays also.
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