Cx3cr1 bv785

Lungs were aseptically removed and placed in ice-cold PBS before processing for flow cytometry. Lungs were then minced and digested using Liberase TM (Sigma-Aldrich) as described previously (54 (link)). ACK lysis buffer was used to lyse the red blood cells, and then total number of viable cells enumerated using trypan blue exclusion. Cell counts were obtained using a TC20 automated cell counter (Bio-Rad). Single-cell suspensions were then stained with Zombie Near-IR (BioLegend) to determine live and dead cells and with the following antibodies for characterization of innate immune populations: CD45 BUV385, CD11b BV510, CD11c–PerCP-Cy5.5, CX3CR1 BV785, SiglecF-PE, CD103 BV711, Ly6C-APC, Ly6G AF700 (BioLegend). Data acquisition was conducted on a Symphony Flow Cytometer (BD Biosciences) and data analyzed using FlowJo 10 (BD Biosciences). The gating scheme used for determining lung cell populations is provided in Supplemental Figure 3.

Flow cytometry data were acquired using a Cytek Aurora equipped with violet (405 nm), blue (488 nm), and red (640 nm) lasers. The following antibodies and stains were used: CCR2-BV421 (clone 475301, 747963; BD), Ly6C-BV510 (clone HK1.4, 128033; Biolegend), B220-BV605 (clone RA3-6B2, 103243; Biolegend), Ly6G-BV711 (clone 1A8, 127643; Biolegend), CD11c-BV785 (clone N418, 117336; Biolegend), CX3CR1-A488 (clone SA011F11, 149021; Biolegend), CX3CR1-BV785 (clone SA011F11, 149029; Biolegend), CD163-PE (clone TNKUPJ, 12-1631-80; Thermo Fisher Scientific), CD64-PE/Cy7 (clone X54-5/7.1, 139313; Biolegend), MRC1-PCP5.5 (clone C068C2, 141716; Biolegend), CD11b-PEFire640 (clone M1/70, 101279; Biolegend) TCRb-APC (clone H57-597, 109212; Biolegend), MHCII-A700 (clone M5/114.15.2, 107622; Biolegend), XCR1-APC/Cy7 (clone ZET, 148223; Biolegend), CD45-APC-Fire810 (clone 30F11, 103173; Biolegend), CD45.1-A647 (clone A20, 110720; Biolegend), CD45.2- BV785 (clone 104, 109839; Biolegend), and LIVE/DEAD Violet dead cell stain kit (L34955; Thermo Fisher Scientific). Flow cytometry data was analyzed using Flowjo 10 software. Dimensionality reduction and cluster identification were performed using the UMAP and Phenograph packages, respectively.