Sfem2 media

The CT26 (BALB/c) murine colon carcinoma cell lines were purchased from and authenticated by the ATCC. MC38 (C57/Bl6) cells were obtained from and authenticated by the NIH. MCA-205 cells were kindly provided by Dr. Andrew Weinberg (Earle A. Chiles Research Institute). CT26 cells were maintained in complete RPMI 1640 (Thermo Fisher Scientific), MC38 cells in DMEM (Thermo Fisher Scientific), and MCA-205 cells in complete RPMI 1640 [10% FBS, 10 mmol/L HEPES, 1% nonessential amino acids, 1% sodium pyruvate (Lonza), and penicillin (100 IU/mL)-streptomycin (100 mg/mL)-glutamine (29.2 mg/mL; Invitrogen)]; the identity of this cell line was verified through monthly assessment of morphology and growth kinetics; in vitro MC38 cells were supplemented with 1% nonessential amino acids and 1% sodium pyruvate (Millipore Sigma). Hs5 cells were obtained from and authenticated by the ATCC, and were grown in SFEM2 media (Stem Cell Technologies) for use in generation of bone marrow-derived macrophages (BMDM). L-929 cells were obtained from and authenticated by the ATCC, and were grown in DMEM þ 10% FBS. All cell lines were used at low passage (<10 passages in vitro after receipt from source). All cell lines were tested and screened negative for Mycoplasma using the MycoAlert test (Lonza).

Primary human bone marrow (AllCells) was obtained from healthy patients and cultured as follows. Briefly, Hs5 cells were cultured for 72 hours in SFEM2 media (Stem Cell Technologies) to obtain Hs5conditioned media. Red blood cells were lysed from the human bone marrow cell population using a hypotonic lysis buffer (BioLegend). The remaining cells were plated in media (75% SFEM2 and 25% Hs5conditioned media) and cultured at 37 C for 72 hours. Flow cytometry was performed to assess the effects of treatment on leukocyte populations of interest.