The activity of MMPs were assessed in the supernatants of serum-deprived DMEM medium from PC3 and Du-145 cell cultures with eNAMPT 100 ng/ml incubation. The MMP activity assay kit (#ab112146, Abcam, Cambridge, MA, USA) was used to analyze the activity of MMPs from cell supernatants by incubating with Green substrate. The fluorescence (Ex/Em=480/520 nm) was measured using a GloMax®-Multi Detection System Fluorometer (Promega, Madison, WI) to calculate the enzymatic activity of MMPs.
Glomax multi detection system fluorometer
Manufactured by Promega
The GloMax®-Multi Detection System Fluorometer is a versatile lab equipment designed for high-performance detection of fluorescent and luminescent signals. It provides accurate and reliable measurements across a range of applications.
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2 protocols using glomax multi detection system fluorometer
1
MMP Activity Assessment in PC3 and Du-145 Cells
2
Measuring GAA Activity in PD Fibroblasts
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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.
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.
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