Human T cell polarization

  1. Isolate naive T cells (see "Human T cell isolation protocol")
  2. Activation and polarization - 4 days culture - day 0
    • Pre-coat plates with anti-hCD3 (1ug/ml; clone OKT3) and anti-hCD28 (2ug/ml; clone 15E8; UCSF core) in PBS. 1-2 hours at 37°C.
    • Culture T cells at 1x106 cells/ml in standard culture medium
      • RPMI 1640 (invitrogen) + 10% FBS, 2mM L-glutamine, 100U/ml penicillin, 100g/ml streptomycin, 1mM sodium pyruvate.
    • Th1 cocktail:
      • rIL-12 (2.5ng/ml; R&D systems)
      • anti-hIL-4 (5ug/ml; clone MP4-25D2; BD pharmingen)
      • anti-hIL-10 (5ug/ml; clone JES3-9D7; Invitrogen)
    • Th2 cocktail: 
      • rIL-4 (12.5ng/ml; R&D)
      • anti-IFNg (5ug/ml; clone B-B1; Invitrogen)
      • anti-IL-10 (5ug/ml; clone JES3-OD7; Invitrogen)
  3. Expansion- 3 days culture- Day 4
    • Resuspend cells at 1x106 cells/ml in a new plate
    • Add rIL-2 (50U/ml; Novartis) + Th1/Th2 cocktails
  4. Re-start above cycle on Day 7 for a total of 14 days in culture.
Category: 
Cell Biology