I am standarizing a protocol to stain Il-17A and IFN-y in PBMCs:

I use plates of 24 wells (CAT Nº 142475) from thermoscientific with a cell concentration of 1million/ml. And stimulate cells with PMA [20ng/ml], Ionomycin [1ug/ml] for 24 h.

Then I apply the fixation/permeabilization Kit from BD [BD Cytofix/Cytoperm TM Plus. Cat. No 555028]. With brefeldin for 4 h. I add 20 ul of IL-17A PE (BD Pharmigen TM Cat. 560486) and 5 ul of IFN-y FITC (BD pharmigen TM cat. 561057).

My results look very good for IFN-y which worked in all the cases I have changed slightly the conditions to see if I was succesful in staining IL-17A. But it was very disappointing when I looked for the IL-17A. I think that the best timing is 24h of stimulation and 4 h of brefeldin

Also, I have tried 48 h for stimulating cells and with bredfeldin for 8h with no results neither.

I see many people having problems with this, do you think that changing the fluorophore could mean any difference?

Any idea of what could improve or do?