4 methylumbelliferyl n acetyl β d glucosaminide substrate

RBL-SX38 cells, which express the human tetrameric form of FcεRI (44 (link)), were routinely cultured in RPMI medium supplemented with 10% FCS, 2 mM l-glutamine, 10 U penicillin/streptomycin, and 50 μg/mL Geneticin (all from Invitrogen) at 37 °C and 5% CO₂ in humidified air. RBL-SX38 cells were seeded at 1 × 10⁴ cells per well in a 96-well plate in RPMI medium supplemented with 10% FCS, 2 mM l-glutamine, and 10 U penicillin/streptomycin and were incubated at 37 °C, 5% CO₂ in humidified air overnight. Following washing with HBSS (Invitrogen)/1% BSA (Sigma-Aldrich), cells were stimulated overnight by incubation with 200 ng/mL IgE diluted in HBSS/1% BSA at 37 °C and 5% CO₂ in humidified air. Cross-linking of IgE-bound FcεRI was carried out by incubation with 50, 500, or 5,000 ng/mL allergen diluted in HBSS/1% BSA for 1 h at 37 °C and 5% CO₂ in humidified air. Supernatants were harvested for analysis. HBSS/1% BSA, polyclonal rabbit anti-human IgE (Dako), and HBSS/1% BSA/0.5% Triton X-100 (Sigma-Aldrich) were used as controls for background, crosslinking, and maximum degranulation, respectively. β-Hexosaminidase release was measured using 4-methylumbelliferyl N-acetyl-β-d-glucosaminide substrate (Sigma-Aldrich) as previously described (45 (link)), and degranulation was expressed as a percentage of the maximum degranulation induced by HBSS/1% BSA/0.5% Triton X-100.