I thawed A20 cells and I had around 70% mortality, because there weren't freeze properly, but that's all I have to work with.
I'm cultivating them in RPMI media supplemented with 10% FBS, 1% L-glutamine, 1% pen/strep, 1% sodium/pyruvate, 0,1% b-mercapto and I added enough glucose to get a concentration of 4500 mg/L.
I've been cultivating them for 6 weeks now, and nothing happens. They grew (slowly) until I had 2 million cells in my flask and it stagnated there. When I count them with trypan blue exclusion, my cells are always alive, but I have around 2 million cells every time, no more. They don't die, but they don't grow either. I tried to transfer them in a bigger volume, then a smaller one, and that changes nothing. I also noticed they tend to precipitate and stick to the bottom of my plate/flask and they are very hard to resuspend, is that normal?
So how do I get these cells to grow? Thank you very much!