2.7 Preparation of bone marrow-derived MDSCs in vitro
Bone marrow cells were isolated from mice by flushing their femurs and tibiae, centrifuging the cell-containing elution (1800 rpm, 5 min), and resuspending them in complete RPMI 1640 (Thermo Fisher Scientific) medium supplemented with IL-6 (40 ng/mL, Peprotech, Suzhou, Jiangsu, China) and GM-CSF (40 ng/mL, Peprotech). The cells were maintained at 37 °C in a 5 % CO2 humidified atmosphere for 4 d. To investigate the effect of IL-35 on the expansion of MDSCs, IL-35, IL-6, and GM-CSF were added to the medium at the same time.