In Vitro Induction, Isolation, and Adoptive Transference of MDSC

BM cells collected from 6- to 8-week-old C57BL/6 mice tibia and femur were flushed with PBS. Red blood cell (RBC) lysis buffer (BD Biosciences; CA, USA) was used to lyse red blood cells. Cells (2 × 10⁶) were cultured at 37°C and 5% CO₂ for 7 days in 6 cm dishes containing 3 ml Roswell Park Memorial Institute (RPMI)-1640 with 10% FBS, 1% streptomycin and penicillin, 1% MEM nonessential amino acids (NEAA) solution, 1% sodium pyruvate (Gibco, NY, USA), 2 μl 2-mercaptoethanol (Sigma-Aldrich, St. Louis, USA), 50 ng/ml GM-CSF (PeproTech), and 40 ng/ml interleukin (IL)-6 (PeproTech). The cells were harvested for testing or sorting by FACS Aria III (BD Biosciences, CA, USA) using CD11b-fluorescein isothiocyanate (FITC) (eBioscience, CA, USA) and Gr-1-PerCP Cy5.5 (eBioscience). Flow cytometry verified that all isolated MDSCs yielded >90% pure population. The purified MDSCs were then adoptively transferred to mice via the tail vein just before UUO. For cell tracing, some UUO mice were injected with MDSCs and stained with CFSE (Invitrogen).