Cathepsin b activity assay kit

Cath B activity was measured using a fluorometric Cathepsin B Activity Assay Kit (Abcam, Berlin, Germany). All cells (1 × 10⁵/well) were seeded in 6-well plates (Sarstedt) and treated with increasing concentrations of nivolumab the following day. After 24 h of incubation, the cells were harvested, washed in PBS (Sigma) and resuspended in chilled Cell Lysis Buffer (4 °C), which was included in the kit, for 30 min, followed by centrifugation at 4 °C for 5 min. The activity was measured in the supernatant according to the manufacturer’s instructions. The output was measured using a Cytation5 reader (Agilent Technologies) at Ex/Em = 400/505 nm.

The activity was measured using a Cathepsin B Activity Assay Kit (ab65300; Abcam) according to the manufacturer’s instructions. Samples were processed without the cathepsin B inhibitor pepstatin A.

The enzyme activity of rFgCatB was measured using Cathepsin B Activity Assay Kit (Abcam, ab65300) according to the manufacturer's instructions. Briefly, 50 μg of rFgCatB protein was adjusted to 50 μL per well with cell lysis buffer for experimental samples in a 96-well plate. Fifty microliters of blank cell lysis buffer were used for measuring background. Next, 50 μL CB Reaction Buffer followed by 2 μL of cathepsin B substrate Ac-RR-AFC (amino-4-trifluoromethyl coumarin) were added to each well. The plates were incubated at 37°C for 2 h protected from light, and fluorescence from the cathepsin B-cleaved substrate was measured at excitation/emission (Ex/Em) = 400/505 nm using a fluorescent microplate reader (Thermo scientific, Varioskan LUX Multimode Microplate Reader). The relative enzyme activity of rFgCatB was represented as the fold increase in the fluorescence intensity compared with the cathepsin B inhibitor-treated control.