Fc block anti cd16 32 clone 2.4g2

Tumors were harvested from donor mice and dissociated using Tissue and Tumor Dissociation Reagent (661563, BD Bioscience, Franklin Lakes, NJ, USA) according to the manufacturer’s instructions. After passing through a 70 μm cell strainer, the single-cell suspensions were pretreated with Fc Block (anti-CD16/32 clone 2.4G2; BioXcell, Lebanon, NH, USA) and stained with FITC-conjugated anti-CD45 (clone 30-F11, BioLegend), PerCP/ Cy5.5-conjugated anti-CD11b (clone M1/70, BioLegend, San Diego, CA, USA), PE/Cy7-conjugated anti-Ly6C (clone HK1.4, BioLegend, San Diego, CA, USA), APC-conjugated anti-F4/80 (clone BM8, BioLegend, San Diego, CA, USA), Pacific Blue-conjugated anti-Gr1 (clone RB6-8C5, BioLegend, San Diego, CA, USA) antibodies, and propidium iodide (P4864, Sigma-Aldrich, St. Louis, Missouri, USA). Each target cell was sorted using SH800S (SONY, Tokyo, Japan). The data were analyzed using the FlowJo software V.10.6.2 (BD Biosciences, Franklin Lakes, NJ, USA).

For murine lung cells, single-cell suspensions were blocked with Fc block (anti-CD16/32, clone: 2.4G2, BioXcell, West Lebanon, NH, USA) and True-Stain Monocyte Blocker (BioLegend), then stained with appropriate antibody mixtures diluted with PBS supplemented with 2% FBS (2% FBS/PBS). After washing with PBS supplemented with 2% FBS/PBS, cells were suspended with 2% FBS/PBS and 0.5 μg/ml propidium iodide. For human lung cells, single-cell suspensions were washed once with PBS and stained with LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific) at 4 ° C for 30 min. Cells were washed once with 2% FBS/PBS; cells were blocked with 2% normal mouse serum and stained with appropriate antibody mixtures diluted with 2% FBS/PBS. After washing with 2% FBS/PBS, cells were suspended with 2% FBS/PBS. Data were collected on a Gallios flow cytometer or a CytoFLEX S flow cytometer and analyzed using FlowJo software v10.6.2 (BD Biosciences). A detailed list of used antibodies is shown in Supplementary Data 2.