Hello everyone, we are struggling to differentiate murine splenocytes population to enrich it with Th17-positive cells. With the protocol described shortly below, we are able to see splenocytes population enrichment with Th17 cells, but the viability is very low (10% top).

CD4+ Cells magnetically separated w/o Fc-blockage

Culture wells coatetd with anti CD3 (5µg/mL) and anti CD28 (5µg/mL)

Culture Medium: IMDM, 10% FBS, 50 µM mercaptoethanol, IL6 50ng/mL, TGF-b 1ng/mL, IL23 5ng/mL, anti IL4 10µg/mL, anti IFN-gamma 10µg/mL

After 4 days of differentation eclls are stimulated for 4hrs with PMA (500ng/mL) and ionomycin (50 ng/mL)

What are we missing? Any help is highly appreciated :)