Tetrazolium Bromide (MTT) assay questionaire. - (Mar/16/2005 )

currently i'm trying to cell viability calculation on my cultured cells. this i tend to apply MTT as an assay for the purpose, according to time intervals. e.g. 24 hours, 48, 72...and so on.

so here, my question is when is the suitable time for me to put MTT in the cells under cultivation by 24 hours time interval?? i'm confusing with some references...that said add the MTT 4 hours before u reach the time required for the test, for example i hav to add MTT at 20 hours and the proceed until 24 hours.

but, some said after reached 24 hours only we add MTT to assay with ELISA reader.

Here, pls give u all's opinions regarding these confusing procedures.

THANKS in advance!

anyone had done this b4?? or anyone can give some opinions and suggestions??

thanks!

I usually add MTT after the incubation period for an additional 30 - 45 min (depends on the cells you have).
for expample: if you are interested in your cells' viability 24 hours after you applied a treatment, wait for 24 hrs (after having added e.g. a cytotoxic agent) and then add MTT (0.5 mg/ml = 1/10 vol, stock 5 mg/ml) directly to the culture medium.
observe the colorimetric conversion (yellow to darkblue) under a light microscope and dissolve the resulting formazan when the cells in your control wells are quantitatively stained (45 minutes should be enough).
aspirate MTT-containing medium and dissolve formazan crystals in DMSO/glycine (6:1) or isoprop....
read absorbance at 565 nm.