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T cells were stimulated with 50 ng/mL phorbol 12-myristate 13-acetate (PMA) and 1 µg/mL ionomycin for 6 hours and then positively selected for their IL-17A expression. After cultivation for 11 days, cells were restimulated with 50 ng/mL PMA and 1 µg/mL ionomycin for 6 hours, followed by an incubation with 1 µg/mL brefeldin A for 4 hours. Cells were then fixed, permeabilized, and stained with Anti-IL-17F antibodies or with the corresponding isotype control antibodies (left images) as well as with Anti-IL-17A antibodies. Flow cytometry was performed using the MACSQuant®
Analyzer. Cell debris and dead cells were excluded from the analysis based on scatter signals.
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