I have worked with HEK cells before and have done freezing and thawing with no problems. I am currently working with HEK 293 T cells to make lentivirus and after thawing them, I can grow them with no trouble in T75 flasks to approximately 80% confluency. However, the problem right now is that after I split the cells in the T75 flasks into 10 cm dishes, I observe a lot of dead cells floating in the media and only a minimum number of cells attached to the surface. The cells that have attached are still clumped together and don't seem to be growing. I have been very gentle with my cells and don't think that it's my protocol that's causing the issue but I could be wrong. I would greatly appreciate any advice.
In addition, I use DMEM 4.5 g/L D Glucose with 10% heat inactivated FBS and 4 mM Glutamax and 0.05% Trypsin with EDTA