Tumor cell killing and cytokine release assays

Prior to use, SW48 cells were harvested in logarithmic phase and diluted to 1 × 10⁵cells/ml in RPMI 1640 supplemented with 5% FBS. 100uL SW48 cells were then added into the wells of 96-well flat bottom plates and incubated overnight at 37° C and 5% CO₂. Next day, 100 nM of various antibodies and controls were added to each well in the presence of 1 × 10⁵ PBMC/50 uL/well for a final reaction volume of 150 uL, and the plates were incubated for 7 days at 37°C and 5% CO₂. 7 days later, 50 uL Cell-Titer Glo reagent (Promega, Cat#G7570) was added to each well and the plates incubated at room temperature for 5–10 min. The plates were then read on a MD SpectraMax i3 for luminescence at 500 ms. For cytokine release assays, 150uL supernatants were collected from each sample well 3 days after incubation at 37 °C and 5% CO_2. Cytokines in the supernatants were detected using Cytometric Bead Array (CBA) Human Th1/Th2 Cytokine kit II (BD, Cat#551809).