I recently tried using NAC on some cell line and wanted to check the viability of cells using MTT. I found that as soon as i add MTT to wells containing NAC it turns bluish black instantly ......some kin dof interaction of the reacting components to me........Then later i read MTT is incompatible in acids and alkalies and therefore tried neutralizing and using the NAC solution still i have got the same results. I had seen in articles that this method was followed and results were obtained. What am I missing here..........could I get some help in this forum. Thanks for anything you could provide.
Regards
Sanjaya
The problem is MTT can be reduced by any nucleophile (pls. read about the rxn chemistry b/w MTT and NADPH) .......so in ur case NAC has a thiol group which is very nucleophilic by itself and therefore reduces the dye even before it enters the cells...so what u can try is ..incubate ur cells with NAC for desired time and then wash it out..after that u incubate the cells with MTT
The problem is MTT can be reduced by any nucleophile (pls. read about the rxn chemistry b/w MTT and NADPH) .......so in ur case NAC has a thiol group which is very nucleophilic by itself and therefore reduces the dye even before it enters the cells...so what u can try is ..incubate ur cells with NAC for desired time and then wash it out..after that u incubate the cells with MTT
Thank you Kavita for your advice, yeah I guess I should have done that, what I could just think of was just the instability of MTT in acids and alkalies and NAC being acidic- I just thought neutralization should help. Certainly I will give it a try.......thanks for the insight.
thanks kavita n sanjaya.. coz i was just about to start similar kind of experiment. i would have ended up with same problm ..
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