Anti fcεr1

Harvested and processed PBMCs were block and stained as follows. For the ILC panel, cells were stained with lineage markers (anti-CD3, anti-CD4, anti-CD8, anti-CD16, anti-CD14, anti-CD19 [eBioscience], anti-CD56, anti-FcεR1, anti-CD11b and anti-CD11c [Biolegend, San Diego, CA]), all labeled with Pacific Blue, anti-CD45-Pacific Orange (Invitrogen), anti-cKit-PerCP-eF710 (eBioscience) and anti-IL-7Rα-APC-Cy7 (eBioscience). Cells were then permeabilized and stained with anti-human IL-13-FITC (eBioscience) and data were collected on a BD FACS Canto II (BD Biosciences).
For the T cell panel, PBMCs were stained with anti-CD3-V500, anti-CD4-Qdot 605 and anti-CD8-PE-Texas Red. Cells were then fixed, permeabilized and stained with anti-IL-4-FITC, anti-IL-2-PerCP, anti-IL-10-Pacific Blue, anti-IL-22-APC, anti-TNF-α, anti-IL-17A-APC-Cy7, anti-IL-5-PE and anti-IFN-γ-PE-Cy7 for 30 minutes. Data were collected on a BD LSRFortessa (BD Biosciences). All flow data were analyzed using FlowJo version 9.4.10.

Mast cells were obtained from the peritoneal cavity of C57BL/6 WT or P2X4^−/− mice by lavage with 5 ml cold PBS + EDTA (2 mM, Gibco). Cells were resuspended in RPMI medium containing 10% FCS, and aliquots were incubated for 10 min at 37°C in the presence or absence of 2 mM ATP (Sigma). Mast cells were identified by flow cytometry as CD11b⁻FcεR1⁺ using anti-CD11b (1/100, BioLegend, clone M1/70) and anti-FcεR1 (1/100, BioLegend, clone MAR-1) antibodies. ATP-induced degranulation of mast cells was evaluated by flow cytometry using antibodies against CD107a (1/100, BioLegend, clone 1D4B) and P2X4-specific monoclonal antibodies and nanobodies in order to detect the externalization of CD107a and P2X4 at the mast cell surface.