Some cell death is inherent in any culture of primary lymphocytes, but almost all the cell dying under these conditions is abnormal.  Do your cells proliferate, which would be expected at this duration of culture?  If the cell proliferate extensively and exhaust the media nutrients (does the media change color?) then they may die.  The first thing worth trying is titrating your stimulating antibodies.  1 ug/mL each of anti-CD3 and anti-CD28 is in the range of concentrations I've always used, but things could be working a little differently with your cells/culture conditions.  I've also never needed to add a "capture" antibody (the anti-hamster Ab); I usually directly coat the plate with anti-CD3 overnight at 4C, wash the plate, block with media, then add the cells in the presence of soluble anti-CD28.  I also don't know why you need to add more anti-CD3/28 at Day 3; the cells should have been adequately stimulated already.  If your cells look fine until Day 3 and then die after you add more anti-CD3/28, then this could be the problem as well.  Good luck!

Hey,

I agree coating the plate with anti-CD3 over night might be the better way to go. I'm not sure if you do but adding 10% FCS to your medium is important (and maybe 0.01 mM 2-ME). For my protocol I add the IL2 from day 0 on but otherwise your protocol seems fine.

Good luck

Hey,

thanks for all the answers so far. I began to titrate the stimulating antibody. I also realize the extra stimulating antibody on day3 might cause the problem because the CD8 T cell grew well at the biginning.

I forgot to mention that the protocol that i used for CD8 T cell cuture is the same that i used for CD4 T cell cuture, which always can gave me very good result of CD4 T cell culture. My quesetion is what is the difference between CD8 T and CD4 T cell culture?

Thank you!

Nicholas Spidale you mentioned blocking coated wells with media, could you tell me how? and for how long? cause I use almost the same protocol as yours but still stuck with the number of cells I got. I coat 24-well directly with anti-cd3, then seed 1-2 million enriched CD8+ T cells in 1ml media with soluble anti-CD28 1ug/ml with either rIL-2 1000U/ml or IL-7 10ng/ml. after 3 days, cells form clusters but don't proliferate.

Shada Elhayek Blocking the wells could be as simple as adding 1 mL media/well (24-well plate) for 30-60 min, but blocking probably isn't a major issue actually. After aspirating the coating antibody, be sure to wash the wells 2 times with 1 mL of PBS.

It's also important to note that most cultures have historically used recombinant human IL-2 for cultures of mouse T cells. Using 100 U/mL of human rIL-2 should be appropriate for most cultures. Perform your anti-CD3/28 + IL-2 stimulation for 48 h, then harvest the cells, pellet, and resuspend in media with IL-2 only. It is probably best to re-adjust the cell density to roughly 1e6/mL at this point, as the cells should begin to expand significantly. Monitor the cell growth and add or exchange media (maintaining 100 U/mL rhIL-2) as needed. Good luck!

Probably you could remove anti-CD3 antibodies during your second stimulation at the step Day 3 and transfer the cells just to the wells with IL2.