T cell activation

Jayakumar, R via methods%40net.bio.net (by R.Jayakumar from roswellpark.org)
Tue Oct 7 11:08:40 EST 2008


 
Wow.. Precoating the plates is good but really not necessary for inducing T-cell proliferation.  Just CD3 coated plates alone should be able to induce T-cell proliferation to a lower level. You can add both anti-CD3 and anti-CD28 as soluble.  Add the required amount of T-cells in each well (50,000-100,000 cells or so), add anti-CD3 (OKT3 clone)and anti-CD28 (clone 28.2 or Mab 9.3) and it should proliferate.  What kind of Mab clones are you working with here (regarding anti-CD3 and anti-CD28)?  No need to supplement with IL-2, since IL-2 induction is what causes T-cell proliferation after you ligate TCR and CD28.  You could also try using a positive control with PMA and calcium ionophore, which will definitely induce T-cell proflieration if your T-cells are viable. 
Jay
 


-----Original Message-----
From: methods-bounces from oat.bio.indiana.edu [mailto:methods-bounces from oat.bio.indiana.edu] On Behalf Of ijpinto from ibmc.up.pt
Sent: Monday, October 06, 2008 3:13 PM
To: methods from magpie.bio.indiana.edu
Subject: T cell activation

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
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