I am trying to differentiate naive CD4+ T cells (isolated from mouse spleen by MACS technique) to Th17 cells.
I covered the plates with anti-CD3 2 μg/ml and anti-CD28 at 1μg/ml the day before and keep them at 4°C overnight.
The next day I add 20 ng/ml IL-6, 3 ng/ml TGFbeta, 5000 ng/ml anti-IFNγ and 5000 ng/ml anti-IL4. (I seeded 1x10^6 cells in 24-well plates in 1 ml T cell medium.)
I incubated the cells at 37°C, 5% CO2 for 5 days. I added 200 μl fresh media (without any reagents) every two days.
At the 5th day I stimulated cells with 10 ng/ml PMA, 1μg/ml anti-CD3 and ionomycin for 4 hours. Then I did FACS analysis with CD4, CD25, CD8 and IL-17A stainings.
Polarization to TH17 cells was unsuccesful as the rates of IL-17A levels were under 40% (94% CD4+, 39% CD25, 0.8% CD8+) while control group without any polarization has 36% IL-17A levels.
Can anybody help me with this issue?
Thank you in advance,