Hi all, I tried to induce th1/2 polarization with human naive CD4 T cells with protocol below.

1. isolate the naive CD4 T cells from commercially provided human PBMC (with MACS)

 2. Seed isolated cells onto CD3(OKT3) pre-coated well at density of 5*10^5cells/ml and

add base media (RPMI1640 with 10% FBS, 2mM glutamax, 50mM 2-ME, 1mM sodium pyruvate, CD28 (CD28.2))

plus, for th1, IL12 (10ng/ml, peprotech) + anti IL-4(5ug/ml, MP4-25D2)

     or for th2, IL4(20ng/ml, peprotech) + anti0IFNr (5ug/ml, AF-285-NA)

3.at day 3 and 5, add base media with IL2(20ng/ml, peprotech) and Th1 or th2 supplement. 

4. subculture cells at day 7 with new cd3 coating dish and maintain the culture as described above.

5. for the FACS analysis, cd3+cd28 (for 5 hrs) or PMA(20ng/ml)+ionomycin(1uM) (for 5 hrs) were treated with monensin (provided in Golgistop kit from BD) and CD4+IFNr or IL4 was labeled.

during maintaining period, typical sphere-like cells are found and they can proliferate well.

my problems are, 

1. I always failed to induce th1 polarization and no IFN-r expression at all.

2. using PMA+Iono combination, IL-4 level was slightly changed; in th2 induction group, the percentage of IL4 positive cells is about 30-35%, while it is 20% and 10-15% in th0(no induction group) and Th1 group respectively. 

--> IL4 positive cell proportion in Th0 and th1 is much higher than expected. Is it normal range? plus, I`m not sure whether th2 induction is fully achieved...

 and using cd3+cd28 combination, I also failed to induce th2 polarization.

3. CD4 expression was almost disappeared with PMA+iono combination.

I don`t know what to do right now....plz help me....

plus, I`m now considering to use ready-to use Th1/2 differentiation kit from STEMCELL. Do you guys think it is helpful? if so, which one should I try? 

Thanks for all.