For experiment in Fig. 1C, 5×104 presensitized MCs were mixed with equal amount of Tregs on microscope slides coated with 0.05 mg/mL poly-L-lysine, and incubated with DNP for 1 or 20 min at 37°C. Cells were fixed with 4% paraformaldehyde for 20 min, washed and mounted with Mowiol. Phase-contrast images were acquired and scoring of the slides was performed in a blinded fashion by evaluating for each condition at least 270 MCs in randomly selected fields from three independent experiments. Nearly, 2×105 presensitized BMMCs were loaded with 1 μM Fura2-AM (Molecular Probes), diluted in 0.5% Pluronic (Molecular

Probes) in DMSO, at room temperature for 10 min. Fura2-loaded BMMCs were plated together with equal number of Tregs onto poly-L-lysine-coated BGB324 purchase coverslips and placed on the stage of an inverted fluorescence microscope (Olympus IX50, Olympus, Japan) equipped with a thermostated chamber (Harvard Apparatus, find more MA, USA), at 37°C. To

analyze (Ca2+)i, a region was drawn around the BMMC and the fluorescence of Fura was used to determine the intracellular levels of free Ca2+ for total 25 min after Ag challenge. The changes in (Ca2+)i are expressed as a ratio of the light emitted at 505 nm upon excitation at the two wavelengths, 340 and 380 nm (F340/F380), after background subtraction. Data were collected using the TillVision Software (Till Photonics GmBH, Germany). Images were analyzed and processed using the ImageJ (Wayne Rasband, NIH; Bethesda, MD, USA) and with custom-made imaging software (Vimmaging, VIMM Padova).

To simultaneously check MC–Treg interactions over the acquisition time, interference contrast (DIC) images were kept at the beginning and at the end of each experiment. DNA ligase Nearly, 4×106 presensitized BMMCs and 4×106 Tregs (ratio 1:1) were challenged with 100 ng/mL DNP-HSA (Sigma-Aldrich) in Tyrode’s buffer/0.05% BSA. After 10 min, cells were fixed by the addition of glutaraldehyde to reach 3% final concentration. Cells were kept for 3 h in 3% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, post-fixed in phosphate-buffered 1% osmium tetroxide for 1.5 h, dehydrated in graded ethanol series and embedded in Epon 812. Thin sections were counterstained with uranyl acetate and lead citrate and examined in a Philips CM 12 electron microscope at 80 kV. Nearly, 1×106 presensitized BMMCs and 1×106 Tregs (ratio 1:1) were challenged with 100 ng/mL DNP-HSA (Sigma-Aldrich) in Tyrode’s buffer/0.05% BSA for 30 min. LTC4 was measured by specific immunoassay (GE Healthcare) while the histamine concentration was determined by ELISA according to the producer’s instruction (DRG Instruments GmbH, Germany). β-Hexosaminidase was determined as previously described 4. For cytokine analysis, equal numbers of IgE-sensitized BMMCs and Tregs were cultured alone or together for 24 h in the presence of 100 ng/mL DNP.