Clone mil4r m1

MMCP-1 in serum samples was measured according to the manufacturer’s instructions (eBioscience). OVA-specific IgE and IgG1 were assessed as previously described, using clones TOε and TOSG1C6 (BioLegend, San Diego, CA) as standards^{24 (link)}. The sandwich ELISA for murine IL-4 was constructed using 11B11 to capture and biotinylated BVD6-24G2 to detect (BioLegend, San Diego, CA) with recombinant protein standard from Shenandoah Biotechnology (Warwick, PA). Murine IL-13 was detected using paired antibodies from Affymetrix/eBioscience (San Diego, CA): eBio13A and biotinylated eBio1316H. Human IL-4 was detected using the antibody pair 8D4-8 and biotinylated MP4-25D2 (BioLegend, San Diego, CA).
Detection of IL-4 production from murine mast cells was enabled by culture in the presence of azide-free anti-IL-4Rα (10μg/ml, clone mIL4R-M1, BD Biosciences, Franklin Lakes, NJ) to block cellular uptake of secreted IL-4. Similarly, IL-4 reuptake by human mast cells was inhibited with anti-IL-4Rα (clone 25463, R&D Systems/biotechne, Minneapolis, MN).

Tregs were depleted from FoxP3^DTR mice by i.p. injection of DT (30 (link)) (50 mg/kg body weight; Sigma-Aldrich) every other day for 5 days (3 injections). Tregs were reduced from FoxP3^DTR+/– mice by i.p. injection of DT (30 mg/kg body weight; Sigma-Aldrich) three times per week for 4 weeks (12 injections). Tissues were harvested on the indicated days as described in Results. Mice were compared to age- and gender-matched DT-treated WT mice or non-DT-treated littermates. In experiments using FoxP3^CreERT2/Gata3^fl/fl mice, Cre recombinase was activated with tamoxifen (Sigma-Aldrich; 2.5 mg i.p. dissolved in corn oil) every 2 days for 14 days starting with concomitant injections of bleomycin (day 0). Mice were treated with bleomycin (Teva, NDC# 00703–3154-01) dissolved in PBS (1 unit/ml) every day for 14 days subcutaneously starting on day 0. In certain experiments, mice were treated with 100μg of anti-CD124 (anti-IL-4Rα) antibody (BD Pharmingen, Clone mIL4R-M1) or isotype (IgG2a, BD Pharmingen, Clone R-35–95) every 2 days for 14 days starting on day 0.