Mast cell degranulation assays

Degranulation was analyzed based on the levels of β-hexosaminidase activity at the supernatant or on CD63 expression on the cell membrane, assessed by flow cytometry, as described in previous studies^23,25. β-hexosaminidase is an enzyme found inside mast cell granules and released at the supernatant after cell degranulation. CD63 is a protein found in the granules and expressed on the cell membrane after degranulation. For each type of essay, we briefly incubated 2 × 10⁴–1 × 10⁵ cells at 37 °C for 30 minutes with several concentrations of the drugs described earlier. For IgE-stimulation, we sensitized mast cells overnight with biotinylated IgE (Abbiotec, San Diego, CA, USA) (0.1 µg/mL), and stimulated them for 30 minutes at 37 °C with streptavidin (0.4 µg/mL) to induce IgE crosslinking. As a positive control for degranulation, we incubated the samples with ionomycin (200 µg/mL) and PMA (10 ng/mL) for 30 minutes at 37 °C.

To carry out the degranulation assays with patient sera, we incubated each serum sample with 1 × 10⁵ mast cells in Tyrode’s buffer for 30 minutes at 37 °C, at a ratio of 1:1, for a total volume of 100 µL. Because of the colour of the serum samples, which interfered with the β-hexosaminidase colorimetric test, only flow cytometry assays with the CD63-APC marker were performed for the sera.

To rule out the drugs’ toxic effects on the cells, a viability count was also carried out in all cases, using trypan blue in the β-hexosaminidase assays and propidium iodide (PI) in the flow cytometry assays.