Clone rm3 1

Cells were dissociated into single cells with 0.05% trypsin (0.1% EDTA) wherever suitable or rinsed off from culture, and resuspended in a FACS washing buffer (PBS with 5% fetal calf serum (FCS) and 2.5 mM EDTA). The cell suspension was then stained with the desired antibodies. The antibodies used in this study were: CD56 (1:50, clone CMSSB; eBioscience), PE-conjugated CD309 (FLK1, 1:50, clone 7D4–6; Biolegend), PE-conjugated CD13(1:50, clone WM15; BD), FITC-conjugated CD31 (1:50, clone WM59; BD), APC-conjugated CD34 (1:50, clone 581; BD), FITC-conjugated CD43 (1:50, clone MEM-59; Biolegend), PE-conjugated CD43 (1:20, clone eBio84-3C1; eBioscience), FITC-conjugated CD45(1:50, clone 5B1; Miltenyi Biotec), FITC-conjugated CD14 (1:50, clone HCD14; Biolegend), PE-conjugated CD68 (1:50, clone Y1/82A; Biolegend), APC-conjugated CD11b (1:50, clone ICRF44; Biolegend), PE-conjugated CD163 (1:50, clone RM3/1; Biolegend), PE-conjugated CD73 (1:50, clone AD2; Biolegend) and APC/Cy7-conjugated CD163 (1:50, clone 12G5; Biolegend). FITC-conjugated mouse IgG2a (1:20, 130-098-846; Miltenyi Biotec), APC-conjugated mouse IgG1 (1:20, 130-098-877; Miltenyi Biotec) and PE-conjugated mouse IgG1κ (1:20, clone P3.6.2.8.1; eBioscience) were used as isotype-matched negative controls. Data were collected with a FACS Calibur flow cytometer (BD) and analyzed using FlowJo software, version 10.0.7.

MDMs were cultured in 12-well plates and exposed to an M1 polarization stimulus using E. coli lipopolysaccharide (LPS) (Sigma, 20 ng/ml) plus recombinant human interferon (rh-IFN)-γ (Fisher, 20 ng/ml) and into M2a using rhIL-4 (20 ng/ml) for 48 h per prior methods (2 (link), 27 (link)). Resident cells without any stimulation were used for baseline comparison. Aliquots incubated in human Fc-γ Block (Biolegend, 422301) for 30 min on ice and then stained using phycoerythrin (PE) anti-human CD80 (Biolegend, 305208, Clone 2D10) and phycoerythrin cyanine tandem conjugate (PE/Cy7) anti-human CD11b (Biolegend, 101216, Clone M1/70) or allophycocyanine (APC) anti-human CD163 (Biolegend, 326510, Clone RM3/1) and allophycocyanin cyanine 7 tandem conjugate (APC/Cy7) anti-human CD206 (Biolegend, 321120, Clone 15-2). Next, FITC anti-human CD68 (Biolegend, 333806, Clone Y1/82A) intracellular staining was performed post-fixation and permeabilization according to the manufacture's recommendations. Stained cells were gated via viable CD11b⁺subsets and identified by CD68CD80 (M1) and CD163CD206 (M2) markers using FlowJo (Tree Star).