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
