Recently, I have conducted a one way mixed lymphocytes reaction to determine allogeneic response. The stimulator cells I used was irradiated at 750cGy (It should be higher within this range: 2000-3000cGy). For responder cells, I didn't culture with cytokines or activating agents such as PHA/ ionomycin. So, I'm not sure whether it is necessary to add cytokines and activating agents? The protocols I searched didn't add cytokines and activating agents. So, I just followed but I can't get any lymphocytes proliferation after pulsed with BrdU. What I get is high dead cell numbers. Does low irradiation level affect the outcome? Is it necessary to wash the cells after irradiated to remove free radical that might kill the cells?
Thanks a lot.