Cells dying before i can get them to in vitro assay - (May/04/2009 )

I've been trying to isolate cells from the spleen and lymph nodes to use for in vitro assays.The organs are ground up using frosted microscope slides in 5 mL of media (1640 RPMI, 10% FBS, 1% ABAM, 50 uM 2-ME). We filter the ground up organs through a nylon mesh, resuspend in 1 mL media and count cells.

It always seems like i get lower cell counts than other ppl in my lab in spite of the fact that i'm pretty sure we do things the same way. Does anyone know why my cells seem to be dying off? We use Trypan blue and count the cells with a hemocytometer. Cells are kept on ice when possible.

If anyone can help me figure out why the cells seem to be dying so soon, let me know.

Thanks!

No real suggestions other than to watch you colleagues do their cell preps one more time and then get them to sit through one with you. There may well be one little thing that you have missed, that they forgot to tell you about.