2.6. β-Hexosaminidase Release Assay

RBL-2H3 cells (1 × 10⁶ cells/well) were seeded in 24-well plates and treated with 200 ng/mL of DNP-specific IgE (Sigma-Aldrich, St. Louis, MO, USA). Cells were treated with or without GBR, GBR-SC61, GBR-SP024 (200 μg/mL), or PP2 (Calbiochem, La Jolla, CA, USA) which is an Src tyrosine kinase inhibitor. After incubation for 30 min at 37 °C, the cells were exposed to 200 ng/mL of DNP-BSA antigen (Sigma-Aldrich) for 15 min at 37 °C. The degree of degranulation was determined by measuring the amount of β-hexosaminidase released, as previously described [13]. The absorbance at 405 nm was measured using a microplate reader (Epoch).