I want to evaluate the CD28 loss on CD4 T cells in culture with different stimuli. I used to stimulate this cells with anti CD3/CD28 dynabeads but actually i don't know if it could interfere with my main goal (CD28 loss).
Most of studies on CD28 loss were developed with feeder cells but nowadays i can't carry out this tests. I will try to use 96 well -plates (U bottom) coated with anti-CD3 (OKT3) and IL-2. Is it enough for cell stimulation?
Thanks in advanced
I used anti-CD3 coated plate to stimulate mouse CD8 and CD8 T cells. It worked perfectly fine. Anti-CD3 alone could induce activation and proliferation in both CD4 and CD8 T cells, just not as efficient as anti-CD3/anti-CD28.
Not sure whether it will work for human T cells.
Thank you very much Zengli Guo . How long do you grow your cell cultures? Do you perform a re-stimulation with CD3 along the culture?
Raúl Villanueva-Romero I examined cell proliferation 3 days and 4 days after stimulation. I used both BrdU and CFSE dilution to check the proliferation. The medium is RPMI1640+10%FBS+1%L-glutamine+1%P/S+ 55uM 2-mercaptoethanol. I did not do re-stimualtion.
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