β-hexosaminidase release assay

Cells incubated in HBSS with or without 10 µM ionomycin and 4 mM of CaCl₂, were placed on ice and cell supernatants were collected. In parallel, cells were lysed with 1% IGEPAL and further diluted 1:5 in dH₂O. β-hexosaminidase activity was determined by incubating cell supernatants or lysates in a 96-well plate with 6 mM of the substrate 4-methylumbelliferyl- N-acetyl-β-d-glucosaminide (4-MU-β-D-GlcNAc; Glycosynth) resuspended in HBSS with 40 mM sodium citrate and 88 mM Na₂PO₄ pH 4.5 for 15 min at 37°C. Fluorescence was measured in a plate spectrofluorimeter Infinite F200 Pro reader (Tecan) at 365 nm excitation and 450 nm emission. The protein content from cell supernatants and lysates was determined simultaneously by using the BCA protein assay kit (Pierce Laboratories) as described by the manufacturer. Absorbance was measured at 560 nm in the same plate reader. HBSS and 1% IGEPAL diluted 1:5 were used as controls. β-hexosaminidase (β-hex) activity was calculated for each sample normalizing to total protein amount as following: β-hex activity in supernatant=(fluorescence (365/450)−HBSS alone)/protein µg. β-hex activity in cell lysate=(fluorescence (365/450)−IGEPAL alone)/protein µg. Total β-hex activity=β-hex activity in supernatant+5× β-hex activity in cell lysate. Finally, the percentage of β-hex release was calculated as following: β-hex release (% of total)=100×(β-hex activity in the supernatant/total β-hex activity).