Hello everyone,
I have established an antigen specific Th1 cell line. I have already re-stimulated them 2 times with irradiated APCs and appropriated antigen.
Now, I am looking for a easier method to re-stimulate my cells excluding the necessity of APCs, antigen.
Can I use PHA, PMA, Con A, soluble anti-CD3/ Anti-CD28 for this purpose?
Thank you!
Hi,
I think restimulating with antigen-charged APC its the cleanest way. APC-T interactions are complex and involve several molecules in both membranes.
If you have any problem with APC, CD3/CD28 should be the proper stimulus because activates TCR signalling. There also exist strategies with MHC-antigen soluble complexes you can check out.
Take account that PMA, PHA, ConA are nonspecific (calcium, PKc pathways) so i do not recommend them for this purpose.
Good luck!
Hi Salina,
when not using APCs I would recommend to coat the anti-CD3/CD28 antibodies to the plastic of your culture dish. In solution without APCs they poorly activate T cells. We commonly used 2h at 37°C in PBS for coating, washed 1-2 times and plated the T cells. I wouldn't use PMA as it massively induces activation-induced cell death. The fact that PMA is not specific for T cells however shouldn't be a problem for you if you're anyway working with a pure T cell culture.
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