Assay for ADAM10 activity

The assay for ADAM10 activity was performed according to the manufacturer’s instructions for the SensoLyte^® 520 ADAM10 Activity Assay Kit (AS-72,226, ANASPEC). Brain tissues or cells were fully homogenized in pre-chilled assay buffer. The supernatant was then obtained by centrifugation at 10,000×g for 15 min at 4℃. An aliquot (50 µL) of supernatant from each sample was then added into the corresponding well of a 96-well microplate with a black, flat bottom plate and non-binding surface. Three replicate wells were used for each sample. A positive control solution (50 µL) containing purified ADAM10 enzyme, or a negative control solution containing only assay buffer, were added to the control wells. A 10 µM 5-FAM reference standard was diluted with assay buffer by 2-fold serial dilution to obtain concentrations of 5, 2.5, 1.25, 0.625, 0.312, 0.156, and 0 µM. Next, 50 µL of these serially diluted 5-FAM reference solutions were added per well. Freshly prepared ADAM10 substrate (5-FAM/QXLTM 520) was diluted 100-fold in pre-chilled assay buffer, and 50 µL was then added to each well to start the enzymatic reaction. The fluorescence intensity at Ex/Em = 490 nm/520 nm was measured immediately and continuously, with the data recorded every 10 min for 2 h. The concentration of enzymatic reaction product was calculated by reference to the 5-FAM fluorescence standard curve, and the data for each sample was plotted as a curve of the relative 5-FAM concentration versus time.