Induction and Isolation of MDSCs

Induction of MDSCs was adapted from previously published work (Alban et al.) (26 (link)). Bone marrow-derived cells from wildtype C57BL/6 mice were prepared as previously described. Cells were then plated at a density of 400,000 cells/cm² and concentration of 1,000 cells/uL in media consisting of 50% complete RPMI (RPMI + 10% FBS + 2mM L-Glutamine) and 50% KR158B conditioned media. Additionally, the media was supplemented with 40ng/mL GM-CSF (R&D 415-ML) and 40ng/mL IL-6 (R&D 406-ML). On day 5, suspended cells were collected, the flask was washed in PBS and scraped using a cell scraper (Fisher), and all contents were joined together in a 50mL conical tube. Cells were collected via centrifugation (4°C, 380 × g, 5 min) and counted by trypan blue exclusion. Cells were then either subjected to flow cytometry (see “Flow Cytometry Analysis”) or utilized for the T cell suppression assay (see “T cell Suppression Assay”).