Hey, guys,

I am using MTT Assay(Mouse 3T3 cell line) to study the cell viability after cultured together with biomedical polymers. I have no much background on cell biology actually. The protocol was referred from publications and a version from this web, which have some difference. Can anyone here clearify my concerns based on failure of my first trail?

1. How much MTT solution needed? What if it is not enough because I noticed normally 10-20ul 5mg/ml MTT solution for 10000 cells/well (seeding cell numbers)?

2. Does it necessary to aspitate the culture media before you add formazan dissolving solution(DMSO)?

3. Because polymer dics (8mm in diameter roughly) were used directly which sit on the bottom of 24 culture plate, my question is did polymer dics effect the growth of cells? The results from the trail showed not good viability after 5 days incubation.

Thanks!

Hi! I've put in the answers to your questions below. In general, with the MTT/XTT family of viable dyes, you have to remember that what the dyes actually measure is the cells metabolic level (in terms of redox potential), which changes depending on culture conditions. With 3T3 cells, ~3-4 h of incubation will be ample if they are not in high-density/confluent states in the wells.
1) --Cell numbers are fine (if your final cell # is between 10-30 k in 24 well plates) - use 10 ul of 2.5 mg/ml for 3-4 h
2) --Very gently. For 3T3, I'd recommend acid-alcohol. Even better, use XTT which does not form the insoluble ppt, but otherwise works in a similar manner.
3) Are your cells growing on the discs? How was the viability of cells cultured alone? Did you have media and media+disc blanks for calibration? 5 days is rather long for 3T3 - are you feeding them? Was the medium yellow even before you added MTT? Did you do a trypan blue cell count before and after 5 days of incubation?
-sam