I badly need your help. I thawed MDA cells (P # 4) and cells looked fine in the T75 flask. I then subcultured after 80% confluency by trypsinization but even after keeping for 15min, all the cells did not come out. I slightly tapped the flask and also scraped the cells to get them out. I plates the cells that were detached and when they grew, half of these seemed to be necrotic. The media I am using is DMEM (high glucose) + FBS (10%) with NEAA, L glutamine and antibiotics. Last time I thawed I had same problem (I did not scratch cells in this one)but my very first stock (that I had purchased) was fine until P # 20 with same media. I am freezing in 70% DMEM + 20% FBS+ 10% DMSO (stepwise freezing).
Edited by Mamcy10, 21 October 2010 - 01:11 PM.