In MTT assay for suspension cells(nalm-6), after drug(dexamethasone) treatment do we directly add MTT in the drug treated wells or should remove drug before MTT addition??
if we should remove drug, how can remove drug in suspension cells?
centrifuge and remove drug before addition MTT ? or use lysine coated 96 well plate ?
Hi Nasrin
I think these ref can be helpful
van de Loosdrecht, A.A., et al. J. Immunol. Methods 174: 311-320, 1994.
Ferrari, M., et al. J. Immunol. Methods 131: 165-172, 1990.
Gerlier, D., and N. Thomasset. J. Immunol. Methods 94: 57-63, 1986.
Alley, M.C., et al. Cancer Res. 48: 589-601, 1988.
Mosmann, T. J. Immunol. Methods 65: 55-63, 1983.
I think there is no need to remove the drug before MTT assay from suspended cells.
Hi, I agree with Rajendra Kumar, it is helpful to test the drug itself in wells without the cell suspension to see if it interacts with MTT (causes a reduction) in order to prevent any interference.
Hello Nasrin,
In most of the protocols it is recommended that you add MTT 4 hrs. before your completion of experiment (dosing treatment), so it generally mean that the drug should stay in wells and removing drug before adding MTT as you have to change media will be a very hectic and cumbersome thing to do and will also incorporate errors in your experiment . So it is not required. Besides, testing the redox potential this time as suggested by Rajendra, next time you can put 3 type of controls in your plate, 1-cells only, media only and 3-Media with drug. And can substract this absorbance further to get original absorbance values. That will avoid any redox testing or complications in future.
Hope it helps !
Dear Nasrin
I have working with suspension cells too. Do you remove the supernatant after 4 hr. MTT incubation by centrifuge? Should I aspirate all the supernatant completely?
saeedeh
we completely remove supernatant by plate centrifuge after 4 hr and then add dmso.
- Badges
- Science topic