Multiparameter Profiling of Immune Cells

THP-1 macrophages and bone marrow-derived macrophages were detached using ice-cold PBS, 0.1% EDTA. Lamina propria cells, peritoneal immune cells and lung immune cells were isolated as described previously.^{13 (link),44 (link),45} The cells were then incubated with FcR blocking antibody (Miltenyi Biotec, Bergisch Gladbach, Germany) for 10 min and stained with anti-CD45-Pacific Blue, anti-CD3-BV650, anti-NK1.1-BV650, anti-B220-BV650, anti-CD11b-BV605, anti-CD11c-PECy7, anti-Ly6C-PerCPCy5.5, anti-F4/80-Fitc, anti-CD64-PE, anti-MHC-II-AF700, anti-CD206-PE-TexasRed, anti-pSTAT6-APC (all from BioLegend, mouse cells) anti-CD124-PE, anti-CD206-APC, (all from Biolegend, human cells) for 15–30 min. ZOMBI-NIR live dead stain (BioLegend, San Diego, CA) was used for discrimination between live and dead cells in all experiments. For pSTAT6 staining, cells were permeabilized using the FoxP3 staining kit from eBioscience. Samples were acquired on an LSRII cytometer (BD, Franklin Lakes, NJ), and analyzed using FlowJo (Tree Star, Inc. Ashland, OR). For RNA expression analysis of lung immune cell susbsets, lung homogenates were sorted on a MoFlo Asterios EQ cell sorter (Beckman Coulter Life Sciences, Krefeld, Germany).