I used normal Isopropanol not acidified one.

DMSO works very well.

Probably you have too much crystals as your cells and very active, try to incubate for shorter period, 1 hour then compare with the 4 hours just to see how much active your cells are. And try to use the  acidified Isopropanol or SDS to dissolve the crystals. usully SDS is better than Isopropanol 

dear Charles K Davis,

I usually using SDS stopper, 100 μL SDS 10% in 0,01 N HCl.

thanks

cheers, dew

I think it is better to test cell cycle duration. may be the cells have short cell cycle so after incubation you have too much cells so 20 ul MTT will turn into formazan crystals and isopropanol is not enough to dissolve them. it is better to reduce the seeding cell number and use 100 ul DMSO or SDS.

I use 200ul of DMSO.  Works really well.

The seeding cell count is 8000/well. Even after 48 hrs the cell confluence will be around 70% in each well. For that density I guess 150ul isopropanol is enough. I seriously doubt that while removing MTT containing media, formazan crystals are also being removed.

I use the Roche kit (11465007001). The stopper is 10% SDS in 0.01M HCl. You add it to MTT containing media  (same volume= 150µl) and has to leave it over night at 37°C to dissolve. This works very well.

Hi

You can add 100 µL of the solubilization solution (0.04 N HCl in isopropanol and or DMSO) into each well  for dissolving of formazan crystals.

Hello everyone , i have been looking at how to make 0.04N HCL in isopropanol, I am wondering if anyone could help out. Thank you