CD3 and CD28 T cell stimulation in TUBES - Technical question (Mar/21/2007 )
Does anybody do a cd3-cd28 stimulation in tubes (eppendorfs)? I have to reproduce experiments that other students did and I am not able to find out the problem. I use the same clone that they used (CD3= clone UCHT1, CD28 = clone 28.2) but I am unable to get phosphorylation of some proteins. Like IKK-beta and IRAK-1. What they did is this :
Stimulation of 4 million of T lymphocytes in 500 ul of RPMI-1640 with 10% serum plus 5 ug/ml of CD3 antibody and plus 5 to 10 ug/ml (I tried both concentrations). All this in an eppendorf tube incubated 15 minutes on ice. I also trie to cross-link the two antibodies by adding about 5 ug/ml of anti-mouse antibody (the both antibodies used previously are monoclonal) also incubated 15 minutes on ice. After, I incubated them the needed time in a water bath at 37 C.
My antibodies are fresh, well stored...
I have also tried to add both antobodies and then incubated directly in the water bath at 37 C. But non of these change anything in protein phosphorylation. Does anybody can confirm or infirm to me the best method I should use to stimulate the T cells with CD3 and CD28 antibodies? I can't use another stimulation method, because I need to continue the work with in the same path!
Thank you very much for your help!
Have a wonderful day!
I never did this experiment, however, if you want to activate with antiCD28 and antiCD3, they have to be coated (on the tube or on beads) to activate, unless anti-CD3 would rather be an inhibitor.
Do you think it's possible to coat the antibody (anti-CD3) in eppendorfd tubes? I thought to stimulate my cells in cd3 coated 6 to 12 wells plates, but it is more difficult to manage because of the different times of stimulation. I can use also the smalest petri dishes coated with anti-CD3 but I find them still too big to stimulate few cells. What is your opinion?
Thank you again for your fast reply!
Have a great day!
I never tried, but why not?
if you still don't see any activation, try to activate with coated beads.
if I remember it's used in this paper : Viola A. Schroeder S. Sakakibara Y. Lanzavecchia A. T lymphocyte costimulation mediated by reorganization of membrane microdomains.[see comment]. [Journal Article. Research Support, Non-U.S. Gov't] Science. 283(5402):680-2, 1999 Jan 29.
Thank you very much again for your help, I will try and I will read the paper that you suggest to me!