Heat inactivated bovine calf serum

786-O (human renal cell adenocarcinoma) cells were maintained in DMEM cell culture medium supplemented with 1% of antibiotics (penicillin/streptomycin, Life Technologies, Carlsbad, California, USA) and 10% heat-inactivated bovine calf serum (Sigma-Aldrich, St. Louis, USA). Immortalized human vascular endothelial cells (ECRF24) were maintained in medium containing 50% DMEM and 50% RPMI-1640 cell culture medium supplemented with GlutaMAX™ (Gibco, Carlsbad, USA) supplemented with antibiotics and bovine calf serum as above. ECRF24 cells were cultured on 0.2% gelatin coated surfaces. Adult human dermal fibroblast (HDFa) cells were maintained in DMEM, supplemented as mentioned above. Human peripheral blood mononuclear cells (PBMC) were freshly isolated as previously described49 .

RAW264.7 cells was obtained from the Bioresource Collection and Research Center and maintained in Dulbecco’s modified Eagle’s medium (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% heat-inactivated bovine calf serum (Sigma-Aldrich, SIAL, USA) with 100 units/ml penicillin, 100 μg/mL streptomycin, and 0.3 mg/mL of L-glutamine (PSG, Thermo Fisher Scientific) and 1 mM sodium pyruvate (Thermo Fisher Scientific) at 37 °C in 5% CO₂ incubator. BMDMs were cultured in RPMI medium 1640 (Thermo Fisher Scientific) supplemented with PSG, non-essential amino acids (NEAA, Thermo Fisher Scientific), 10% fetal bovine serum (Thermo Fisher Scientific), and 20 ng/mL M-CSF (R&D Systems, MN, USA). CD4⁺ T cells and stimulated BMDMs were cultured in RPMI medium 1640 supplemented with PSG, NEAA, 10% fetal bovine serum, and mIL-2 (20 ng/mL, Peprotech, Rocky Hill, USA). For detection of IL-2, mIL-2 was removed from the complete medium.