Lymphocyte culture trouble!! - (Apr/03/2013 )

Hey everyone,
I am trying to culture splenic lymphocytes for estimation of cytokines in supernatants after 48 hrs of culture. The method is well standardized in our lab and I had no problems until now. Recently, a few of my experiments were complete disaster as after 48 hrs of culturing I could not detect any cells. After centrifugation, I used to get a thick white pellet of cells indicating good cell proliferation but nothing as such is happening now, I've changed media, serum but nothing works. Any insight into this is seriously appreciated. Thanks in advance.

make sure that you isolated the splenocytes correctly in the first place. 48 h of ex vivo culture wont cause death of all splenocytes generally, maybe just a portion. I suppose the problem is in your isolation procedures.