Th1/Th2 polarization.

Jianzhu Chen Lab @MIT

1) Coat a 24 well plate with 1 ml 5 mg/ml anti-CD3 antibody and 5 mg/ml anti-CD28 antibody containing PBS per well over night at 4-degrees. Remove liquid from wells immediately prior to use.

2) To each well add 10⁶ naïve CD4 T-cells in 1 ml T cell medium (10% FCS, Pen/Strep, L-Glu, 2-ME RPMI.) For Th1 polarization also add 5 ng/ml IL-12 and 10 mg/ml anti-IL-4 antibody. For Th2 Polarization add 25 ng/ml IL-4 and 10 mg/ml anti-IFN-gamma antibody. Incubate at 37-degrees.

3) After 24 hours of stimulation add IL-2 to a final concentration of 40 U/ml.

4) After 72 hours of stimulation, remove the cells from stimulation, split them 1:3, and maintain for another 72 hours in the presence of IL-2 plus polarizing cytokines. At this point analysis can be performed.