T cell activation

ijpinto from ibmc.up.pt via methods%40net.bio.net (by ijpinto from ibmc.up.pt)
Mon Oct 6 14:12:46 EST 2008


Dear all,

I'm having a problem in inducing proliferation of CD4+ and CD8+ cells in 
vitro. I've coated 96 well plates with 10 micrograms/ml anti-CD3 (OKT3) 
and added 2 micrograms/ml soluble anti-CD28. After 4 days of incubation I 
still don't get any proliferation. These same conditions worked well for 
PBMCs. My specific questions are:

1- Do I have to perform any special treatment of the plates before adding 
the anti-CD3 antibody? I'm just adding the antibody, incubating 3 hours at 
37ºC, washing with HBSS and adding the cells right afterwards. However, I 
saw some studies where they add goat anti-mouse IgG prior to the OKT3. 
Does this makes an essential difference? 

2- Do I have to supplement with IL-2, in addition to anti-CD3 and 
anti-CD28, to get T cell proliferation?

Your help is welcomed. Thanks a lot!

Jorge


Jorge P Pinto, PhD
Iron Genes and Immune System
Cell Culture and Genotyping (Head)
Institute for Molecular and Cell Biology
Rua do Campo Alegre, 823
4150-180 Porto
Portugal
Phone: +351-226074956
email: ijpinto from ibmc.up.pt


More information about the Methods mailing list