I performed MTT assay for which I cultured lymphocytes in a 96-well plate. I incubated the cells (lymphocytes) with my test substance for 24 hours, after which I added 2ul of MTT reagent to each well. After incubating with MTT reagent for 4 hours, 100ul of DMSO was added, after 30 minutes OD was recorded at 595nm. the problem is that I used 8 concentrations from 50 to 400ug/ml, the 9th column is normal cells that weren't incubated with test substance, but I didn't observe a gradient from light purple to dark purple(from higher concentration to lower concentration). all the wells were equally purple, the cell viability seems to be uniform irrespective of the dose/concentration. my LD50 came out to be 163.46. I'm attaching pictures for reference.
The concentration of the test substance that you are taking, might be too low to induce any inhibition in cell proliferation. Try considering a higher range of test substance to get a better picture of the cytotoxicity.
How was your IC-50 calculated in this case?
Dear Mehar Khalil,
We had this problem in our lab too. We changed every substance in our work. And we found that reason was the drug that we used in our work. I think you should find the IC50 of your substance at first. If you found it before and you didn't get the result you should find the IC50 again. On the other hand, it is very important to culture same amount of cells in your wells.
Good luck.
Ali Khalafizadeh sir I have checked the OD value of each well using an Elisa plate reader. I’m attaching the picture for data, can I estimate the IC50 using this data ?
Mehar Khalil If you used different amounts of your substances for example from low to high concentration, can be possible. But I think you should use more variable amounts of your substances to get more differences among your ODs to find more valuable IC50 for your work. But at first, calculate the IC50 with these data. This is a link for calculating IC50 with Prism GraphPad software if you don't know the way: https://youtu.be/CD9CZjzDTEE
Good luck.
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