Anti mhc 2 af700

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).

On the day of euthanization, one ear per mouse was harvested, followed by mechanical disruption. Tissue was digested under rotation in DMEM (41965-039, Gibco) at 37 °C with 4 mg/mL collagenase IV (17104019, Gibco). Tissue was filtered using 40 μm strainers and resuspended in FACS buffer (1% FBS, 1 mmol/L EDTA, and 0.02% NaN₃ in PBS) to achieve a single-cell suspension. Samples were treated with anti-CD16/CD32 (101302, BioLegend, San Diego, California, USA, 1:100) for 20 min on ice followed by antibody staining. To analyze immune cell populations, cells were stained with anti CD45-PacificBlue (103216, Biolegend, 1:400), anti CD4-APC (100412, Biolegend, 1:400), anti γδ-TCR-PerCp AF700 (100714, eBioscience, San Diego, CA, USA, 1:400), anti CD8-APC/Cy7 (46-5711-82, Biolegend, 1:400), anti CD11b-BV605 (101257, Biolegend, 1:200), anti F4/80-PE (12-4801-82, eBioscience, 1:200), anti Ly6G-FITC (551460, BD Biosciences, 1:400), anti MHCII-AF700 (107622, Biolegend, 1:800), anti CD11c-PE/Cy7 (117318, Biolegend, 1:400), anti Foxp3-PE/eFlour610 (61-5773-82, Thermo Fisher, 1:200) and Zombie-Aqua (423102, Biolegend, 1:800) on ice for 20 min. Data were acquired on a CytoFLEX S flow cytometer (B96621, Beckman Coulter, Brea, CA, USA) using FACS Diva 6.1.3 and analyzed using FlowJo v10.5.3 (BD Biosciences).