I use trypsin to harvest my cells (NIH 3T3), but they don't detach after trypsin incubation. I tried to increase trypsin volume and incubation time in 37C incubator. I usually use 3ml of trypsin per 25cm2 T flask and incubate for 3 mins at 37C. I got a very small amount of cells and subcultured cells also don't detach from the bottom of the flask. Has anyone encountered the same problem? How did you solve it?