I seeded 1x105 cells in each well and incubated for 24hrs and the following day 100 µl of plant samples with different concentrations and 100 µl of complete growth media (CGM) (90%DMEM+10%FBS) were added into the wells and incubated for another 24hrs.
Then on the 3rd day, media was pipetted out and 100 µl of CGM and 20 µl of MTT solution was added into each wells and after 4 hrs incubation 100 µl of DMSO was added and was read at 517nm.
Hello Anjana
In MTT assay MTT is reduced to formazan crystal which is purple in color by the metabolic active cells by the action of oxidoreductase enzymes in the mitochondria. So when the purple formazan crystals are solubilized by DMSO the end point color remains purple and not yellow as you mentioned.
MTT won't be reduced to formazan crystal if dead cells are present in the well in which case the color may be yellow.
Meaning your extract killed the cells in all concentration. You can have a look in microscope with high power magnification before incubate with MTT. See whether there is morphology changes. Even 96 wells can use microscope to have a quick look.
Try to reduce the extract concentration accordingly to obtain IC50
Mavis Lim Thank you for your response. No my extract did not kill the cells which is why I think it did not change from purple to yellow/colorless as expected results and all wells remained in purple color. I don't know why my extract did not kill the cells.
I'm so sorry, I'm mixing up the color conversion.
You may try to double up the plant extracts concentration, but keep in mind that the solvent have to be less than or equal to 0.1%.
I did experienced that my CRUDE extract cannot extend cytotoxicity towards cancer cell lines even up to 1mg/mL. Coz crude extract (especially methanol crude extract) have a lots of nutrients and antioxidants, which may protect the cells. If using low polar solvent for crude extraction, then killing effect maybe obvious.
Sorry once again
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