Mixed lymphocytes reaction (MLR) - (Oct/01/2014 )

Hi all,

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.

Regards,

Tan

Taiwan

Im not familiar with MLRs but i have worked with irradiated splenocytes as stimulator cells and responders as t cells. I ve used 400rads of radiation but i also have to pulse the splenocytes with peptide. I wash the peptide off the cells before starting the co-culture and have always had proliferation of t cells. 

So you could try washing your stimulator cells before adding responders or you might have to add a peptide for lymphocyte recognition and response!

Good luck!