CIK and CIK-Tregdel cells were generated from PBMCs and PBMCs depleted of CD4 +CD25+ regulatory T cells, respectively.
Ficoll-separated cells were cultured in XVIVO 20 serum-free medium (Cambrex, USA), consisting of 100 U/ml of recombinant human IL-1a, 50 ng/ml of anti-CD3 antibody (e-Bioscience, USA), and 1000 U/ml of recombinant human IFN-? (PEPROTECH, USA) at 37? in a humidified atmosphere of 5% CO2 for 24 h before 300 U/ml of recombinant human Interleukin (IL)-2 (PEPROTECH, USA) was added to media.
Cells were subcultured every 3 days in IL-2 and IFN-? containing medium. On day 14, the phenotype and cytotoxicity of CIK cells were assayed.