Arvo mx light 1420 multilabel luminescence counter

Cathepsin activity was assayed fluorometrically with Z-Arg-Arg-MCA (Peptide Institute, 3123-v) for CTSB and Z-Phe-Arg-MCA for CTSL (Peptide Institute, 3095-v), as previously reported.⁵⁷ (link) Briefly, WAT or cell pellets were resuspended in lysis buffer (352 mM KH₂PO₄, 48 mM Na₂HPO₄, 4 mM EDTA, pH 6.0) and incubated on ice for 60 min before centrifugation for 10 min at 2,100 ×g. Supernatant was collected and protein concentrations were determined with a BCA kit (Pierce, 23225). Supernatant was then added to the reaction buffer (4 mM DTT in lysis buffer) as an assay buffer.
CTSB activity. A total of 100 μL assay buffer (containing 1 μg of protein) was mixed with 100 μL substrate buffer (10 μM Z-Arg-Arg-AMC diluted in 0.1% Brij 35; Sigma, B4184) and incubated at 37°C for 30 min. Fluorescence was measured using a Wallac ARVO MX/Light 1420 Multilabel/Luminescence Counter (PerkinElmer, Waltham, MA, USA) with an excitation/emission of 360/460 nm.
CTSL activity. A total of 100 μL assay buffer (containing 10 μg of protein) was mixed with 100 μL substrate buffer (10 μM Z-Phe-Arg-AMC and 10 μM CA074) and incubated at 37°C for 30 min. Fluorescence was measured as described above.