Evaluating BMDC Activation by R837

BMDCs obtained from C57BL/6 mice were co-cultured with R837, NS (negative control) or LPS (positive control) as previously described [20 (link)]. Briefly, BMDCs were obtained from the femur and tibia of C57BL/6 mice, and cultured with 20 ng mL⁻¹ rmGM-CSF (Xiamen Amoytop Biotech Co., Ltd., China) and 10 ng mL⁻¹ rmIL-4 (Pepro Tech, USA). The medium was replaced three-quarters every 2–3 days until adherent cells were collected on the 8th day. The harvested immature DCs were resuspended in RPMI 1640 medium without cytokines and co-cultured with R837, NS or LPS for 48 h. Then incubated with 1 µL FITC-anti-mouse CD11c monoclonal antibody, PE-anti-mouse CD86 monoclonal antibody, and APC-anti-mouse CD80 monoclonal antibody (Biolegend, USA) for 30 min before evaluated by flow cytometry (Beckmann, BD Bioscience, USA).