Hello everybody,

I wish to establish a protocol for expansion and activation of  murine CDT8+ T cells in spleen of vaccinated mice.

I am trying to do it by co-culturing specific tumor antigens (whole apoptosis tumor cells) with splenocytes from the vaccinated tumor bearing C57BL/6 mice.

here, C57/BL6 mice were inoculated with 5*10ex6 MC-38 cell lines, and when tumor mass was established, these tumor bearing mice were vaccinated with bone marrow derived dendritic cells (DCs) pulsed tumor antigen (whole apoptotic MC-38 cells).

After 4 times of DCs pulsed tumor antigen vaccination to tumor bearing mice, I sacrificed these vaccinated mice and collected the spleen (My purpose to check memory CD8 T cells in spleen of vaccinated mice), and then, I co-cultured these splenocytes with gamma irradiated MC-38 cells (whole apoptosis MC-38 cells) for 5 days in RPMI 10% FBS in the presence of murine IL-2, but after co-culturing 3 days to 4 days, almost splenocytes cells were died, and the color of media was very yellow.

I have tried to harvest cells after 2 day co-cultured  but splenocytes still reduced.

so please help me!

Thank you in advanced!