3.4. Degranulation Assay and MTT Cell Viability Assay in Mast Cells

The mucosal mast-cell-derived rat basophilic leukemia cells (RBL-2H3) were purchased from Bioresource Collection and Research Center (Hsin-Chu, Taiwan). The cells were cultured in DMEM containing 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin in 10 cm cell culture dishes at 37 °C in a humidified chamber with 5% CO₂ in air. The level of degranulation in RBL-2H3 cells was evaluated using β-hexosaminidase release assay induced by A23187 or antigen as reported before with some modifications [40]. Briefly, the cells were seeded in a 96-well plate (2 × 10⁴ cells/well, for the A23187-induced assay) or a 48-well plate (3 × 10⁴ cells/well, for the antigen-induced assay) overnight. The cells for the antigen-induced assay were sensitized with anti-DNP IgE (0.5 μg/mL; Sigma) during seeding overnight. RBL-2H3 cells were then treated with the samples (0.5, 5, and 50 μM) for 30 min in Tyrode’s buffer with a maximal DMSO dose of 0.5%. For the A23187-induced assay, the cells were activated by addition of A23187 (final concentration 0.5 μM), while cells for the antigen-induced assay were activated by the addition of DNP-BSA (final concentration 100 ng/mL) for 30 min. Azelastine (10 μM) served as the positive control. The amount of β-hexosaminidase was detected using the method utilizing p-NAG as the substrate according to the procedure described before [41].

The viability of the RBL-2H3 cells in the presence of the samples (10 and 100 μM) was determined using the methylthiazole tetrazolium (MTT) assay according to a previous method [41].