I want to use MTT test for lymphocyte cells that are the suspension, but I also need the supernatant for Eliza. Does anyone have a protocol to help? I did this by DMSO but I didn't observe any blue color
Dear Hamidreza,
As suggested by Weber, you can centrifuge the cells and collect the supernatant. However, I would suggest centrifuging the supernatant once again at full speed to remove cell debris before using it for ELISA. You can also store the supernatant at -80oC and proceed with the cells first for you MTT assay.
I do not think you have to use DMSO at any step. Correct me if I am wrong.
I hope this information helpful. Goodluck
I did the same your comments and got a superfluous fluid, but then I did not know how to do the MTT test on compressed cells.
@ Hamidreza,
You need to resuspend the pellet (your compressed cells) in fresh media then proceed with MTT assay as in the following link.
http://www.abcam.com/kits/mtt-assay-protocol
Hi!
It is so easy.
1. Sedimentate cells using centrifuge and use supernatant for ELISA. If concentration of studied protein is too low, use filters or cold vacuum evaporation for its concentration increasing.
2. Add fresh medium with MTT reagent to cells. Incubate for approx. 3 h (or other period in agreement with your previous experience) on shaeker (gently!) at appropriable temperature.
3. Sedimintate cells using centrifuge. Remove medium. Add fixed volume DMSO.
Good luck.
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