I need to treat bone marrow derived macrophages with a specific peptide for studying the antigen presentation. In the same experiment, I need to measure intracellular cytokine release and do CFSE experiments. I was wondering if bone marrow derived macrophages are the feasible cells for this experiment.
Dear Tabasum
Macrophages are terminally matured cells specialized in phagocytosing and destroying intracellular/intravesicular pathogenic agents. This characteristic show us that they already had phagocytized the antigens.
Monocytes are their precursors and they are better for antigen presentation than more differentiated cells, due to the fact that they are circulating cells, express higher levels of MHC II and are really avid to phagocyte any Ag and migrate to regional lymphnodes to present Ag to CD4+ T cells.
You can do the peptide presentation and afterwards measure cytokines and do CFSE from T cells. If you want to do intracellular cytokines of monocytes it is important to remind that these cells fluoresce spontaneously and it is sometimes difficult to quantify IC cytokines, produced in low quantities, even after effective Golgi blockade.
Please remember that CFSE is very toxic and must be used in very low concentrations (1-2 nM). I would suggest to change CFSE for PKH26, that is less toxic and very effective to quantify cell divisions.
Good luck,
Dewton
Dear Tabasum
Macrophages are terminally matured cells specialized in phagocytosing and destroying intracellular/intravesicular pathogenic agents. This characteristic show us that they already had phagocytized the antigens.
Monocytes are their precursors and they are better for antigen presentation than more differentiated cells, due to the fact that they are circulating cells, express higher levels of MHC II and are really avid to phagocyte any Ag and migrate to regional lymphnodes to present Ag to CD4+ T cells.
You can do the peptide presentation and afterwards measure cytokines and do CFSE from T cells. If you want to do intracellular cytokines of monocytes it is important to remind that these cells fluoresce spontaneously and it is sometimes difficult to quantify IC cytokines, produced in low quantities, even after effective Golgi blockade.
Please remember that CFSE is very toxic and must be used in very low concentrations (1-2 nM). I would suggest to change CFSE for PKH26, that is less toxic and very effective to quantify cell divisions.
Good luck,
Dewton
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