Fitc anti fcεri

After stimulation with rmIL-33 and/or rmSCF (both 50 ng/mL) (Peprotec), the BMMCs were washed with PBA (PBS supplemented with 5 mg/mL BSA and 10 mM NaN₃). Afterwards, we added anti−CD16/CD32 and rat-IgG (Jackson) to prevent non-specific binding. MCs were stained with PE−anti-CD117, FITC−anti-FcεRI, and PECy7−anti-IL-33R (all Biolegend). To investigate the activation of p65/RelA in p65/RelA−eGFP MC/9 reporter MCs after stimulation, we determined the eGFP expression by flow cytometry. The cells were analyzed with an LSR II flow cytometer (BD), and the data were analyzed with FlowJo 10 (Treestar Inc., Ashland, OR, USA).

BMMCs were harvested and washed with PBA (PBS supplemented with 5 mg/mL BSA and 10 mM NaN₃). BMMCs were treated with anti‐CD16/CD32 and rat‐IgG (Jackson) to prevent non‐specific binding of the staining antibodies. BMMCs were stained with PE‐anti‐CD117 or APC‐anti‐CD117, FITC‐anti‐FcεRI or PE‐CD107α (all Biolegend). MC/9‐NFAT and MC/9‐NF‐κB MC lines were stained with PE‐anti‐CD117. All cells were analysed with a LSR II flow cytometer (BD). Data were analysed with FlowJo 10 (Treestar Inc., Ashland, OR).

BMMCs were harvested and washed with PBA‐E (PBS, 5 mg/ml, BSA, 10 mM NaN3, 2 mM EDTA). To block unspecific binding prior to antibody staining, BMMCs were preincubated in the presence of rat IgG (Jackson) at 4°C for 5 min. For the determination of the purity of the BMMC cultures, cells were stained with PE‐anti‐CD117 (Biolegend) and FITC‐anti‐FcεRI (Biolegend). Furthermore, we used FITC‐anti‐CD117 (Biolegend), APC‐Cy7‐anti‐CD117 (Biolegend) and PE‐anti‐CD275/ICOS‐L (Biolegend) as indicated in the figures and figure legends. All antibodies were diluted in PBA‐E and were incubated at 4°C. After 20 min, BMMCs were washed with PBA‐E and analysed by using LSR II flow cytometer (BD). For the analysis of T_reg/BMMC cocultures, we used the FoxP3 Transcription Factor Staining Buffer Set (Thermo Fisher). According to the manufacturer's protocol, cells were washed and fixed and were intracellularly stained with APC‐Cy7‐anti‐CD4 (Thermo Fisher), PacBlue‐anti‐FoxP3 (Thermo Fisher), PE‐anti‐RORγt (Thermo Fisher) and PerCP‐eFluor 710‐anti‐HELIOS (Thermo Fisher). Subsequently, cells were analysed with the LSR II flow cytometer (BD) and data were analysed by using FlowJo 10 (Treestar Inc., Ashland, OR).