I am inducing EAE in mice by adoptive transfer from female donor mice. The Hoocke protocol I follow states that I should be able to harvest enough CD4 Th1 from each donor mouse to inject in 3 recepient mice. I need 10 million T cells per mouse but I can't harvest more than 2 to 3 million from 1 donor. I induce myelin autoreactive T cells in donor mice for 10 days. I harvest spleen and Lymph node, filter total cells and culture them in vitro 72hours with MOG, anti-CD3, IL-12, IL-2 and IFNg. I isolate CD4 T cells using Dynabeads kit and count alive T cells with trypan blue. Do you guys have any idea what is going wrong with my in vitro culture? Could you please give me your protocol for this? Thank you
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