Cell Stimulation With Polyinosinic: Polycytidylic Acid (Poly I:C) and LPS

CIK cells were cultivated in 6-well plate for 12 h and then transfected with 2 μg/ml poly I:C or immediately stimulated with 25 μg/ml LPS or added PBS as control. The concentrations chosen for each stimulant were deemed optimal from previous studies (26, 27). Cell samples were washed with PBS for three times and harvested at 0, 6, 12, 24, 48, and 72 h after stimulation. For Western blot, the samples were lysed in Lysis buffer containing protease inhibitors. As for the gene expression analysis, samples were harvested with Trizol reagent. Freshly prepared HKLs, seeded in 24-well plate (1 × 10⁶ cells/ well), were stimulated with 50 μg/ml Poly I:C (Sigma-Aldrich) or 25 μg/ml LPS (Sigma-Aldrich) or PBS as control for 3, 6, 12, 24, 48, and 72 h. The stimulants were diluted in complete medium before addition to the cells. The treatments were terminated by dissolving the cells in TRIzol reagent.