Degranulation of basophils (RBL-2H3) determined by β-hexosaminidase release

The enzymatic activity of β-hexosaminidase that is contained in the cell’s granules was monitored. The cells were seeded in 96-well tissue-culture plates at 1 × 10⁵ cells in 100 μL of Eagle’s minimal essential medium with Earle’s salt (MEM) supplemented with 10% fetal bovine serum, and incubated at 37 °C in 5% CO₂ for 5 h. Then sensitization of the cells with IgE was performed by the addition of 50 μL of the mAb 2F5 (2 μg/mL) in MEM per/well and the plate was incubated for 12 hours at 37 °C in 5% CO₂. The monolayers were washed three times with Tyrode’s buffer to eliminate the mAb 2F5 excess. Allergens were added to the washed monolayers at different concentration to get the maximum degranulation. As positive controls for the cell’s FcεRI-mediated degranulation, we stimulated them with either a mouse monoclonal anti-FcεRI (F4) antibody⁵⁰ (in non-sensitized cells) or with a Goat anti-mouse IgE H&L when the mAb 2F5 was already bound to the cells. Total β-hexoaminidase activity was determined in wells where the cells were treated with lysis buffer (Tris-HCl 50 mM, 150 mM NaCl, and 1% Triton X-100 to pH 7.5 with cocktail of proteases inhibitors). The rZea m 12 was used as a negative control. After the treatments indicated for each experiment, secretion was allowed to proceed for 30 min at 37 °C. From each well, three aliquots of 25 μL were transferred to a separate plate and 50 μL of the substrate solution (1.3 mg/mL p-nitrophenyl-N-acetyl-β-D-glucosamine in 0.1 M citrate, pH 4.5) was added before incubating the plates for 90 min at 37 °C. The reaction was stopped with 150 μL of 0.2 M glycine (pH 10.4), after which the absorbance was measured at 405 nm. The results are expressed as a percentage of the total β-hexosaminidase activity present in the cells. The spontaneous release observed in the absence of stimulus was subtracted from each experimental value.