4 methylumbelliferyl α d glucopyranoside 4mu

To study the rhGAA uptake and correction of GAA activity in PD fibroblasts, the cells were incubated with 50 µM rhGAA for 24 h, in the absence or in the presence of 10 mM L-CAR. Untreated cells were used for comparison. After the incubation, the cells were harvested by trypsinization and disrupted by 5 cycles of freezing and thawing.
GAA activity was assayed by using the fluorogenic substrate 4-methylumbelliferyl-α-D-glucopyranoside (4MU) (Sigma–Aldrich) according to a published procedure³¹ (link). Briefly, 25 µg of cell homogenates were incubated with the fluorogenic substrate (2 mM) in 0.2 M acetate buffer, pH 4.0, for 60 min in incubation mixtures of 100 µl. The reaction was stopped by adding 1 ml of glycine-carbonate buffer, 0.5 M, pH 10.7. Fluorescence was read at 365 nm (excitation) and 450 nm (emission) on a Promega GloMax Multidetection system fluorometer. Protein concentration in cell homogenates was measured by the Lowry assay.

NSCs were assayed for GAA enzyme activity. Cells were dissociated using Accutase and incubated in a lysis buffer containing 50 mM Tris pH 7.5, 100 mM NaCl, 1% Triton X-100, and protease inhibitors (cOmplete mini tablets, 4693159001, Sigma-Aldrich, St. Louis, MO, USA) for 30 min on ice. After centrifugation, supernatant was collected for assays. Protein concentrations were measured with a BCA assay. The GAA activity assay was adapted from [26 (link)]. Briefly, 15 µL of the cell lysates with 10 µg total proteins were incubated with 40 µL of 2.2 mM fluorogenic substrate 4-methylumbelliferyl-α-D-glucopyranoside (4MU, Sigma-Aldrich, St. Louis, MO, USA) in 200 mM sodium acetate buffer, pH 3.8, plus 5 µL of 960 µM acarbose (Sigma-Aldrich, St. Louis, MO, USA) in ddH₂O at 37 °C for 1 h in a 96-well plate. Reactions were terminated by adding 200 µL of 150 mM EDTA, pH 11.4. Fluorescence was measured at 360 nm (excitation) and 465 nm (emission) on a CLARIOstar microplate reader (BMG LABTECH).