I am wondering why i am seeing the absorbance (from pink color) at the last step of MTT assay in the wells where there are no living cells (checked with microscope). Colour of MTT reagent looks fine(green) which indicates that there are no contamination in MTT.
MTT reagent act on Mitochondria. If the cell death may be due to apoptosis the cells still respond to MTT. Try to do MTT immediately after your experiment,and carry out another set MTT after 24 hours. If MTT response is different then try to find out mode of cell death in your assay.
Are you using an MTT assay kit? If so, which one?
I have only ever seen yellow MTT solution not green.
I found this in the ATCC MTT assay kit handbook:
"If the MTT Reagent is bluegreen, do not use and refer to the troubleshooting guide on page 6" http://www.atcc.org/~/media/DA5285A1F52C414E864C966FD78C9A79.ashx
In my experience you should get approx 0.05 AU at 585 nm for medium only controls.
If you're getting higher AU than this, and you MTT is green I'd say there is something wrong with your MTT.
i agree to James. there is something wrong with your MTT reagen.
good luck
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