T cell proliferation assay - (Jul/18/2006 )

Hello Members,

I am at the moment doing Proliferation Assay using CD4-positive T cells. I pulse the cells after 48 hrs with 3H-Thymidine to check for proliferation levels. I am having a huge problem at the moment...cells that are not stimulated with anti-CD3 and anti-CD28 are giving me high CPM values...in the range of 8000-10000. whats worse is that without adding anti-CD28, proliferation levels are much higher than co-culturing the T cells with both anti-CD28 and anti-CD3. Please...someone help me out with this. what could i possibly do?

Thanks

-loislane81-

QUOTE (loislane81 @ Jul 18 2006, 12:18 AM)

May be uneven cell density in each well.

-Minnie Mouse-

are you triggering too much your cells? are they dying?

-Missele-

QUOTE (loislane81 @ Jul 18 2006, 05:18 AM)

Hi,
Before daring to give an answer, and provided your "odd" results are reproducible (something uneven cell density would not allow between experiments), it would be important to know:
1) The source of CD4+ T cells.
2) The method of isolation.
3) The clone of anti-CD28 and anti-CD28 (some are stimulatory but some are inhibitory).

Hope this helps. Regards,
Gerardo

-Gerardo-