I want to check my stimulated monocytes ability to present antigen to CD4+ T cells.
Also wondering if I analyze the result on FACS, do I need to irradiate the monocytes?
Any tips and inputs are highly appreciated.
Yes, Ioana, it is possible. Stimulator cells (monocytes) should be irradiated or treated by mitomycin C. MLR in response to syngeneic monocytes should be served as control. In several days you can determine 3H-thymidine incorporation or analyse results by FACS. Also, you can use preliminary staining of CD4 T cells with CFSE to determine the number of cell divisions by FACS. Obviously, difference between allogeneic and syngeneic cultures is very clear. Under microscope you will see many blast cells in cultures stimulated by allogeneic stimulator cells. You can find detailed protocols easily. Good luck!
Yes, Ioana, it is possible. Stimulator cells (monocytes) should be irradiated or treated by mitomycin C. MLR in response to syngeneic monocytes should be served as control. In several days you can determine 3H-thymidine incorporation or analyse results by FACS. Also, you can use preliminary staining of CD4 T cells with CFSE to determine the number of cell divisions by FACS. Obviously, difference between allogeneic and syngeneic cultures is very clear. Under microscope you will see many blast cells in cultures stimulated by allogeneic stimulator cells. You can find detailed protocols easily. Good luck!
You should be able to get away without irradiating the monocytes which save you hassle. For old 3H-proliferative assay you do.
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