I'd done MTT assay on a cancer cell line to study cytotoxicity of crude plant extracts. The incubation period for the assay were 24, 48 and 72 hours. However, i found that some of the ic 50 results were not time dependent. I'd read in the journal that the ic 50 value is smaller as the incubation period increase, eg; 24h: 50ug/mL, 48: 45 ug/mL and 72: 40 ug/mL. But some of my result, the increasing incubation period gave higher ic 50 value, eg :24hr: 24 ug/mL, 48: 45 ug/mL, 72: 40 (ug/mL) ( The trend was quite like this). All the crudes are from the same plant source, except it were extracted from different solvents. I'd repeated the assays many times and if the results were not valid, what is the problems involved actually.
Thanks. my workflow: i seed the cell at identical numbers but at the same time for 24, 48 and 72 hours incubation period then add the plant extracts the next day (after the cells reach more than 50 % confluency) and incubate for 24, 48 and 72 hours. So, for MTT i do at different timeline for each incubation hours with the same MTT procedures you had mention.
Dear Ashikin,
MTT assays and related assays (i.e. MTS) rely on a mitochondrial reductase to convert the tetrazole to formazan. The assumption is that the conversion is dependent on the number of viable cells...but there is always the possibility that treatment of your cells may result with increased enzymatic activity without actually having an effect on cell number or cell viability. That is why performing your MTT experiment along with simple cell counts (Trypan blue exclusion assay) is a good idea to complement your observations.
There may be two other possibilities:
1- Some compounds has an enhancing effect on cell proliferation when it is prepared only within certain ranges of concentrations
2- Your compound may activate cell metabolism.
Suggestions:
1. Increase the concentration of compound
2. Do the MTT with empty compound
Hope this helps !
As far as i understood, you do 3 MTT experiments (3 different plates), u start them at the same time but you end them at different time points, and for each of the three plates u used different plant extract samples as the drug (extracted in different solvents).. couldn't that be what's causing the differences in metabolism/viability (opposing the expected increasing incubation decreasing IC50) ..the solvent?
I would like to ask on this raised issue (@Himanshi): Is it a common practice to change (media+drug) everyday for drug incubations that are more than 24 hours?
I leave the same (media+drug) for the drug incubation period, but I am interested to know how this affects the cells.. Unlike w/ cells grown in flasks (large numbers of cells), I don't see any media color change in the 3 days of drug incubation in the small wells, so why should we consider changing media and risk losing cells?
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