β-Hexosaminidase release assays

LAD 2 cells were plated in a 96-well V bottomed plate in imaging buffer and incubated at 37 °C for 10 min. Antagonists were then added as indicated and incubated with the cells at 37 °C for 5–10 min before the addition of agonists. Cells were then incubated at 37 °C for an additional 20 min before being centrifuged (2500RPM, 10 min, 4 °C) and the supernatants removed. Supernatants were incubated with substrate (2 mM 4-nitrophenyl N-acetyl-β-D-glucosaminide diluted in 0.2 M citrate buffer) for 2 h at 37 °C. The reaction was stopped by the addition of Tris-HCl (1 M, pH 9.0) and absorbance at 405 nm measured (Expert Plus Microplate reader, Biochrom Ltd). Spontaneous β-hexosaminidase release was determined by the addition of imaging buffer only. Total β-hexosaminidase content was determined by the addition of Triton X-100 (0.06 %) to lyse the cells. Background readings were determined for later subtraction from wells containing only release buffer and substrate. All test conditions were done in duplicate in a single experiment and each experiment repeated a minimum of three times. Average background values and average spontaneous β-hexosaminidase release values were subtracted from the reading for each well. Release was then expressed as a percentage of the average total β-hexosaminidase content determined from the Triton-treated wells. Results are displayed as mean ± SEM. For all statistical data comparisons, percentage values were log10 converted to transform the data before a one-way ANOVA with post hoc Tukey test was performed. In all figures, * = p < 0.05, ** = p < 0.01. The LDH cytotoxicity assays were conducted using a kit (Roche) as per the manufacturer’s instructions.