Mast cell degranulation assay

Mast cell degranulation was analyzed as described previously [40]. BMMCs from WT or Rock1−/− mice were washed and starved in RPMI containing 0.5% BSA for 6 hours and then resuspended in Tyrode buffer (10 mM HEPES buffer, pH 7.4, 130 mM NaCI, 5 mM KCI, 1.4 mM CaCI₂, 1 mM MgCI₂, 5.6 mM glucose, and 0.1% BSA). For measuring degranulation in response to IgE, cells were sensitized with 10 mg/mL anti-dinitrophenyl (anti-DNP) IgE mAb SPE-7 (Sigma-Aldrich) for 1 hour, washed twice with 23°C Tyrode buffer, equilibrated in Tyrode buffer to 37°C for 5 minutes, and then treated for 15 minutes with DNP-human serum albumin (Sigma-Aldrich). The degree of degranulation was determined by measuring the release of β-hexosaminidase.