In vitro CD4 Th0, Th1, Th2, Th17 and Th22 cell polarizations
@-CD3 coated (clone: 145.2C11) (1μg/ml) + @-CD28 coated (clone: 37.51) )(1μg/ml)in PBS OVN 4oC in 50ul for 96 wells/plates.
Use 200ul in 96 wells/plates U bottom plates for a total of 50.000 pure CD4 T cells –> Culture for 5 days!
Th0
Coated anti-CD3/CD28
Th1
Coated anti-CD3/CD28
+ IL-12 (5ng/ml)
+ anti-IL-4 (clone: 11B11) (10μg/ml)
Th2
Coated anti-CD3/CD28
+ IL-4 (10ng/ml)
+ anti-IFN-γ (clone: XMG1.2) (10μg/ml)
Th17
Coated anti-CD3/CD28
+ TGF-β (2ng/ml) + IL-6 (50ng/ml) + IL-1β (20ng/ml) + IL-21 (100ng/ml) + IL-23 (20ng/ml)
+ anti-IL-4 (clone: 11B11) (10μg/ml) + anti-IFN-γ (clone: XMG1.2) (10μg/ml)
Th22
Coated anti-CD3/CD28
IL-6 (50ng/ml) + IL-23 (50ng/ml)
+ anti-IL-4 (clone: 11B11) (10μg/ml) + anti-IFN-γ (clone: XMG1.2) (10μg/ml)
Day 3 look at the cells and add some fresh medium if really yellow.
Day 5 PMA-Iono restim and intracellular FACS staining.
In vitro CD4 Th0, Th1, Th2, Th17 and Th22 cell polarizations —> PDF