Mccoy medium

The four human colon cancer cell lines HT-29, HCT-116, SW480, and SW620 were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). HT-29 and HCT-116 cells were cultured in McCoy Medium (ATCC) supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin/streptomycin (pen/strep). SW480 and SW620 cells were maintained in RPMI 1640 medium (ATCC) containing 10% (v/v) FBS and 1% (v/v) pen/strep. All cell lines were incubated at 37°C in 5% CO₂. To induce CD137L signaling cell culture plates were coated with 10 µg/ml recombinant human CD137-Fc (R&D Systems, Minneapolis, MN, USA) at 4°C overnight. To preserve cell surface epitopes cells were then carefully detached using accutase solution (Sigma-Aldrich, St. Louis, MO, USA) and seeded in the pre-coated wells in a defined number.