I have been trying to detect IL-4 (CD124) and IL-15a receptor expression on human monocytes and U937 cells via flow cytometry without any success. I differentiate the monocytes into DCs with GM-CSF and IL-4; and IL-4-stimulated U937 have activated STAT6 but no IL-4 or IL-15Ra receptors detectable on either cell type.
U937 cells were serum-starved overnight in the same manner that I do for detection of activated STAT6. CD14+ monocytes are positively selected from PBMCs and have been cultured in overnight with or without GM-CSF to see if GM-CSF helps up-regulate the IL-4 receptor.
Cells are blocked on ice for 30 mins, then stained with commercial Abs for 30 min, ice, dark, and finally fixed before analyzing the cells...
Does anyone have any experience with this or have any advice?
Thank you!