Cd206

The MACS CD206⁺ enriched population of lung resident macrophages were incubated with FcR Block (422302; BioLegend) for 5 min and stained at a dilution of 1:50 with one of two panels of directly conjugated antibodies for 30 min at 4°C: anti-human CD45 (563792; BD), CD204 (371906; BioLegend), CD206 (321132; BioLegend), CD14 (562698; BD Biosciences), CD16 (302028; BioLegend), ACE2 (FAB933P; R&D), HLA-DR (307618; BioLegend), CD11b (393114; BioLegend), CD11c (301644; BioLegend); anti-human CD45 (324016; BioLegend), CD204 (371904; BioLegend), and CD206 (321103; BioLegend). Stained cells were then washed with FACS buffer (2% FBS in PBS) three times, and then incubated with cell viability marker propidium iodide (PI, 1 μg/ml, 421301; BioLegend). Flow cytometry was performed on a FACS Aria II (BD Biosciences).
Living (PI⁻) single, immune (CD45⁺), and lung resident macrophages (CD206⁺) were stained for the above panel of cell surface antigens that have previously been suggested to segregate them into AMs and IMs, and that were differentially expressed according to the scRNA-seq transcriptomic profiles obtained from lung slice culture and sorted into CD206⁺CD204^hi and CD206⁺CD204^lo populations. The sorted populations were directly subjected to 10x single-cell mRNA sequencing at BSL2 as described above, which confirmed their molecular identities as AMs and IMs, respectively.

Plasma-EVs, aged RBC-EVs, or EV subtypes were stained with PKH26 Red Fluorescent Cell Linker Dye (Sigma-Aldrich). EVs were washed twice with 10% exosome-free FBS in RPMI to quench the unbound dye. Recombinant annexin V (BD Biosciences) or functional grade blocking monoclonal antibodies against phosphatidylserine (PS) (Millipore), CD36 (Stemcell Technologies), CD163, CD206 (BioLegend), TLR1 (Invivogen), TLR2, and TLR4 (BioLegend) were used at multiple concentrations (0.01–2.0 µg/mL) to block EV–monocyte binding. In some experiments, EVs were incubated with annexin V or anti-PS antibody. In other experiments PBMCs were incubated with monoclonal antibodies against CD36, CD163, CD206, TLR1, TLR2, or TLR4 in a 5% CO₂ incubator at 37°C for 1 h. PBMCs (500,000) were cultured with EVs, in a final volume of 0.5 mL for 24 h. PBMCs were stained with CD14-PerCP/Cy5.5 (BioLegend) and were fixed in 2% paraformaldehyde solution. PBMCs were subject to flow cytometry, and percent binding of monocytes to EVs was measured by gating on CD14⁺ cells.