Fc block 2.4g2

To detect cytokines, BALF was collected and stored at −80°C. IL‐1β was quantified by ELISA according to the manufacturer's instructions (R&D Systems). Concentrations of IL‐6, moncoyte chemoattractant protein (MCP)‐1, IFN‐γ, IL‐10, IL‐12p70 and TNF‐α proteins were determined by cytokine bead array mouse inflammation kit (Becton Dickinson). For flow cytometric analysis, BALF cells were treated with red blood cell lysis buffer (Merck), and cell numbers and viability were assessed via Trypan blue exclusion using a haemocytometer. BALF cells were incubated with Fc block (2.4G2; eBiosciences), followed by staining with fluorochrome‐conjugated monoclonal antibodies to Ly6C, Ly6G, CD11c and I‐Ab (MHC‐II; BD Biosciences, Franklin Lakes, USA). Neutrophils (Ly6G⁺), airway macrophages (CD11c⁺ I‐Ab^low), dendritic cells (DC; CD11c⁺ I‐Ab^high) and inflammatory macrophages (Ly6G⁻ Ly6C⁺) were quantified by flow cytometry, as described previously.¹⁵ Live cells (propidium iodide negative) were analysed using a BD FACS Canto II flow cytometer (BD Biosciences) and FlowJo software (FlowJo, Irvine, USA). Total cell counts were calculated from viable cell counts performed via trypan blue exclusion.