β-Hexosaminidase Release Assay

The β-hexosaminidase release assay was performed following previously described (Gao et al., 2017). For the measurement of C48/80-induced β-hexosaminidase release, LAD2 cells or MPMCs were pretreated with FRE (200–600 μg/ml) at 37°C for 30 min followed by adding C48/80 (10 μg/ml) for a further 1.5 h incubation. Thirty microliters of supernatant were collected and mixed with 50 μl of substrate solution (0.57 mg/ml 4-Methylumbelliferyl N-acetyl-β-D-glucosaminide in 0.133 M sodium citrate buffer, pH 4.3) in a 96-well black flat bottom plate at 25°C for 2 h and the reaction was terminated by adding stop buffer (50 mM glycine and 5 mM EDTA·Na₂, pH 10.5). The fluorescence intensity was read at λ _ex 355 nm/λ _em 460 nm by a fluorescence microplate analyzer (Thermo Scientific Fluoroskan Ascent FL, United States).

For IgE/FcεRI-mediated β-hexosaminidase release, 1% anti-SP serum sensitized-RBL-2H3 cells were pretreated with FRE for 30 min at 37°C and then co-incubated with SP (40 ng/ml) for another 1.5 h. The supernatant was collected for the β-hexosaminidase detection.