2.8. β-Hexosaminidase Secretion Assay

The degranulation response of RBL-2H3 cells was quantified by measuring the level of β-hexosaminidase released in the supernatants [20]. RBL-2H3 cells were grown on 6-well plates at a density of 2 × 10⁵ cells for 24 h. The cells were then treated with 800 ng/mL DNP-specific IgE (Sigma-Aldrich) overnight. To remove the excess IgE, the cells were washed twice with Tyrodes’ assay buffer (pH 7.3). The cells were then stimulated with DNP-BSA (Invitrogen), suspended in 500 µL of extracellular buffer with 0.1% BSA, and incubated at 37 °C for 1 h. After incubation, 50 µL of the supernatant was incubated with 200 µL of 1 mM p-nitrophenyl-N-acetyl-β-D-glucosaminide (Sigma-Aldrich) in 0.05 M citrate buffer (pH 4.5) at 37 °C for 1 h. The enzyme reaction was terminated by adding 500 µL of sodium carbonate buffer. The OD of each reaction was recorded at 405 nm. The percentage of viable cells was calculated using the following equation (Equation (3)):

where, Al is the absorbance of the cell lysate and Am is the absorbance of the medium.