Fitc il 4

Human basophils were gated on FcεRIα-FITC/CD123-PerCP/Cy5.5/CD203c-PE (BioLegend, San Diego, CA, USA) positive cells after extracellular and intracellular staining. The expression levels of CD203c-PE, CD62L-APC, FcεRIα-FITC, CCR7-APC, CD63-APC, IL-13-APC, B cell-activating factor (BAFF)-APC (BioLegend, San Diego, CA, USA), IL-4-PE-Cy7, and IL-6-APC (eBioscience, San Diego, CA, USA) in basophils were quantified and expressed as relative fluorescence units (the ratio of mean fluorescence intensity normalized to controls) or as a positive percentage of total basophils.
Mouse basophils were gated on CD49b-APC/IgE-PE (BioLegend, San Diego, CA, USA). The expression levels of the activation marker CD200R-FITC (BioLegend, San Diego, CA, USA) (26 (link)), IL-4-FITC, and IL-6-FITC (eBioscience, San Diego, CA, USA) were quantified and expressed in the same way as for human basophils. A FACScanto™ Π flow cytometer (Becton Dickinson, San Jose, CA, USA) and Lysys II software (Becton Dickinson, San Jose, CA, USA) or FlowJo Software (Tree Star, San Carlos, CA, USA) were used to acquire and analyze the data.

The MNCs from lungs were obtained and resuspended in the fluorescence activated cell sorting (FACS) buffer (1‐2 × 10⁶ cells/mL), and then blocked using anti‐CD16/CD32 antibody (clone 2.4G2; BD Biosciences, San Diego, CA) to avoid non‐specific binding and subsequently labelled with isotype controls or the following antibodies: FITC‐CD4, FITC‐IFN‐γ, FITC‐IL‐4 and PeCy7‐FoxP3, PeCy5‐TCR‐β (eBioscience) and PE‐PBS‐57/mCD1d tetramer (gifted by the Natural Institutes of Health tetramer core facility). Invariant natural killer T cells were determined as PBS‐57/mCD1d tetramer and TCR‐β double‐positive cells. For intracellular cytokine staining, lung MNCs were cultured for 4‐6 hours with 50 ng/mL phorbol 12‐myristate 13‐acetate (PMA) and 500 ng/mL ionomycin in the presence of monensin (1 μL/mL) (all from Sigma). Cells were collected and washed, and then intracellular staining was performed following the manufacturer's procedures (eBioscience). Treg cells were measured as CD4⁺ FoxP3⁺ cells. Intracellular staining for FoxP3 was performed using Fix/Perm buffer reagents (eBioscience) according to the manufacturer's protocol. Cells were detected by flow cytometry (BD FACSAria III, BD Biosciences) and the acquired data were analysed using FlowJo software (Tree Star, San Carlos, CA).