Isolation and Expansion of Myeloid-Derived Suppressor Cells and Macrophages

Primary bone marrow-derived MDSCs and MΦs were prepared from C57BL/6, MyD88^−/−, or TLR2^−/− mice as previously described (19 (link), 22 (link), 56 (link)). MDSCs were expanded for 4 days in RPMI-1640 supplemented with 10% fetal bovine serum (FBS), 1% l-glutamine, 1% HEPES, 1% antibiotic-antimitotic, 50 μM beta-mercaptoethanol, 40 ng/ml GM-CSF, and 40 ng/ml G-CSF with 40 ng/ml IL-6 added at day 3 of culture. Following expansion, MDSCs were purified using an anti-Ly6G microbead kit (Miltenyi Biotec). MΦs were propagated for 7 days in RPMI-1640 supplemented with 10% FBS, 1% l-glutamine, 1% HEPES, 1% antibiotic-antimitotic, 50 μM beta-mercaptoethanol, and 10% conditioned medium from L929 fibroblasts as a source of macrophage colony-stimulating factor (M-CSF) (28 (link), 57 (link)). For visualizing MΦ invasion into biofilm by confocal microscopy, MΦs were stained with CellTracker deep red (1 μM; Invitrogen) according to the manufacturer’s instructions.
Human monocytes were obtained from healthy human donors by the UNMC Elutriation Core Facility by countercurrent centrifugal elutriation, in full compliance and with approval of the Institutional Review Board (IRB). Cells were cultured at 1 × 10⁶ cells/ml in RPMI-1640 supplemented with recombinant human M-CSF, 10% human serum, and 1% antibiotic-antimitotic for 7 days until harvest for experiments.