Fibronectin coated glass coverslips

The TIRF assay was performed using WGA-488–loaded BMMCs sensitized with DNP-specific IgE overnight. Cells were allowed to attach to fibronectin-coated glass coverslips (10 µg/ml; Sigma-Aldrich) before stimulation with 20 ng/ml DNP-HSA. TIRF images were then acquired for 15 min (exposure time of 200 ms). During the observation, the cells were cultured at 37°C in a 5% CO₂ atmosphere. Fluorescence data were acquired with a TIRF imaging system (Eclipse Ti-E; Nikon). Images were acquired with a QuantEM 512 SC camera (Roper Technologies) and NIS-Elements AR software (version 3.1; Nikon). Image sets were processed with ImageJ software. A WGA-containing granule was defined by a minimum of a square of 3 × 3 pixels (1 pixel = 0.16 µm) of strong intensity signal, orange to white intensity from the pseudocolor scale.

BMMCs were sensitized with DNP-specific IgE overnight, plated onto fibronectin-coated glass coverslips (10 µg/ml, Sigma Aldrich) for 45 min at 37°C with 1 mM MnCl₂, and then activated with 20 ng/ml DNP-HSA for the indicated times. BMMCs were fixed by incubation for 15 min on ice in 3.7% wt/vol PFA and then for a further 10 min in NH₄Cl (50 mM in PBS). The cells were then incubated for 1 h with specific primary antibodies in permeabilization buffer (PBS with 1 mg/ml bovine serum albumin and 0.05% wt/vol saponin; Sigma-Aldrich), washed twice, and incubated for another hour with fluorescent-conjugated secondary antibody in permeabilization buffer. Lastly, the cells were mounted on slides in Prolong gold antifade reagent in the presence or absence of DAPI (Invitrogen). Confocal microscopy was performed with an LSM 700 system (ZEISS) and 63× NA 1.4 objective. The images were processed with the LSM Image Browser (ZEISS) and ImageJ software (version 1.43; National Institutes of Health).