Fluorogenic Assay for Cathepsin-B Activity

As previously described by our laboratory [23 (link)], to determine total and B-specific cathepsin activities the following assay components were mixed in a 96-well plate using PBS as diluent: first WLL fluid (50 μL), 2 μg Z-LR-AMC (fluorogenic Peptide Substrate, R & D systems, Minneapolis, MN, USA) ± 66 μM inhibitor (Z-Phe-Phe-FMK, MBL International, Woburn, MA, USA) in a total volume of 150 μL. The assays samples were incubated at 37°C for 1 h then fluorescence was measured using a plate reader at 380 nm excitation and 460 nm emission. Cathepsin-B specific activity was calculated as follows: relative fluorescence units (RFU) from assay without inhibitor minus the assay with inhibitor.