1640 media

BM-MDSCs were cultured as previously reported.²⁰ Briefly, BM was collected from mouse femurs and treated with red blood cell lysis buffer (BioLegend, San Diego, CA, USA). Isolated BM immune cells were then washed with 10% fetal bovine serum (FBS) and filtered through a 40-µm strainer (Greiner Bio-One, Kremsmünster, Austria). Thereafter, 3 × 10⁶ cells were resuspended in 14 mL of culture medium: 500 mL Roswell Park Memorial Institute-1640 media (Lonza, Basel, Switzerland) supplemented with 55 mL FBS (Atlanta Biologicals, Flowery Branch, GA, USA), 2 µL 2-mercaptoethanol, 12.5 mL 1 M HEPES (Thermo Fisher Scientific, Waltham, MA, USA), 500 mg streptomycin sulfate (Meiji Seika, Chuo City, Tokyo Japan), and 5 mL 100 mM sodium pyruvate (Sigma-Aldrich, St. Louis, MO, USA). To generate BM-MDSCs, interleukin (IL)-6 (Peprotech, Rocky Hill, NJ, USA) and granulocyte macrophage-colony stimulating factor (GM-CSF; Peprotech) were added, each at 40 ng/mL. The BM-derived cells were cultured in an incubator at 37°C, 5% CO₂, and 95% humidity for four days for further ex vivo or in vivo studies.