Cxcr2 sa044g4

Mouse BAL cells were treated with α-CD16/32 Fc block (eBioscience) and mouse serum (Thermo Fisher Scientific) prior to staining with antibodies. Relevant full minus one (FMO) samples for each group were used as controls. Antibodies used were as follows: Ly6G (1A8, BioLegend), CD11a (M17/4, BioLegend), CD11b (M1/70, BioLegend), CXCR2 (SA044G4, BioLegend), and CXCR1 (FAB8628A-025, RnD). Live cells were gated following staining with DAPI (Invitrogen) prior to acquisition. BAL neutrophils were gated according to Ly6G⁺ and forward scatter (FSC)/side scatter (SSC) properties. Cells were acquired on an LSRFortessa (BD). Compensation was performed using BD FACSDiva software and data analyzed with FlowJo version 10.

Flow cytometric analysis was performed as previously described (16 (link)). Cells were labeled with fixable viability dye (eFluor506 or eFluor780; eBioscience), blocked with anti-CD16/32 (clone 2.4G2), and stained with fluorochrome-conjugated antibodies specific for CD11b (clone M1/70), Ly6G (1A8), CD45 (30-F11), IL4ra (mIL4R-M1), and CD14 (rmC5-3), all purchased from BD Pharmingen. Fluorochrome-conjugated antibodies specific for CD101 (polyclonal), and CD62L (MEL-14) were purchased from eBiosciences. CXCR2 (SA044G4) was purchased from Biolegend. For intracellular staining, cells were fixed and permeabilized with BD cytofix and cytoperm solutions, then stained with fluorescent antibodies specific for CD206 (MR6F3; eBiosciences). Flow cytometry was performed with a FACS Symphony A3 cell analyzer (BD Biosciences). Cells were gated on forward and side scatter after doublet exclusion and analyzed on FlowJo v10 software (Supplemental Figure 1A).