I have performed an MTT assay using A549 cells against different concentrations of my drug betacyanin (0.625 mg/mL, 1.25 mg/mL, 2.5 mg/mL, 5 mg/mL, 7.5 mg/mL, 10 mg/mL, 20 mg/mL and 30 mg/mL) and I used Milli-Q water as my control and DMSO as my blank. Once I calculated the percentage of cell viability and plotted the respective graph; all my values were above 100%. Does anyone have any idea why this may be?
Hello Vindya
In MTT assay MTT is reduced to formazan crystals by the activity of NAD(P)H dependent oxidoreductase enzymes present in the viable cells. There is a possibility that the drug (betacyanin) which you are using may have an increased effect on the mitochondrial enzyme activity without causing an increase in cell viability. Another possibility I could think of is that the drug may not be having any toxic effect on the cells because of which you could have got 100% viability.
What I would suggest is that in addition to MTT assay you do a cell count using trypan blue for comparison. You would get an idea on cell viability for both these assays.
Good Luck.
Did you use freshly prepared MTT ?
Did you refer to any previous results of the same drug with MTT ?
It will be good to include positive control of drug that induces cell death as your treatment might affect the mitochondrial function (assayed by MTT). In addition, you should pay attention that the DMSO concentration
of the control and your treatments is identical and not exceed 0.1%. Furthermore, you can measure cell number by counting
rather than MTT.
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