Ab65300

Assay of cathepsin B (CTSB) and cathepsin D (CTSD) activities was carried out using CTSB assay kit (abcam, ab65300) and CTSD assay kit (abcam, ab65302) according to the manufacture's protocol. Briefly, 50 μg of cytosolic fraction protein was added up to the final reactant to 200 μL per well in a 96‐well plate. Next, samples were treated with 140 mm site‐specific substrates in assay buffer at 37 °C with 10 mm DTT for 0.5 h and then analysed by flow cytometer.

Enzyme activity assays for cathepsin B (cat# ab65300, Abcam), cathepsin D (cat# ab65302, Abcam), and cathepsin G (cat# ab126780, Abcam) were performed on three snap-frozen MDOTSCC samples of the cohort of six patients used for NanoString mRNA analysis, according to the manufacturer’s protocol. A snap-frozen tonsil sample was used as an appropriate positive control for the cathepsins B (17 (link)) and D (18 (link)) assays, and a denatured sample of the same tonsil was used as an appropriate negative control. The capthepsin G assay kit was supplied with its positive and negative controls. The results were obtained using the Variskan Flash plate reader (Thermo Fisher Scientific). Experiments were performed in duplicates with averages taken for each.

Brain cathepsin B activity was measured 2 h after trauma using a fluorometric assay kit, as described by the manufacturer (ab65300; Abcam, Cambridge, MA, USA). Briefly, tissues were washed twice in ice-cold phosphate-buffered saline and then homogenized in extraction buffer, as described by the manufacturer. After 10-min incubation on ice, the extract was centrifuged at 10,000 g for 5 min, and 50 μL of supernatant was mixed with an equal volume of 2 × reaction buffer and 2 μL of substrate in a 96-well microplate. Plates were kept in the dark at 37 °C for 1 h, and fluorescence was recorded using a FLUOstar Optima plate reader (BMG LABTECH GmbH, Ortenberg, Germany). Protein concentration was determined by the bicinchoninic acid assay method (Bio-Rad, Hercules, CA, USA). Cathepsin B activity was measured in triplicate and was expressed as fluorescent units/mg of protein. For the determination of enzyme activity, we isolated the region of trauma for analysis.

Lysosomal enzyme activity in iPS-RPE cells cultured in 12-well plates was assessed with the kits for the following enzymes: cathepsin D (Abcam, ab65302), cathepsin B (Abcam, ab65300), acidic sphingomyelinase (Abcam, ab190554), glucosylceramidase (Abcam, ab273339) and LysoLive lysosomal acid lipase (Abcam, ab253380). Assays were performed following the manufacturer's protocols. For the cathepsin D, cathepsin B, acidic sphingomyelinase and glucosylceramidase assays, whole-cell protein lysates were used with results normalized to the protein concentration determined by Bradford assays (Bio-Rad).

The enzymatic activities of cathepsin B, D, and K were measured using a fluorometric assay kit from Abcam (ab65300, ab65302, and ab65303, respectively), according to the manufacturer’s instructions. Briefly, C2C12 cells were washed twice in ice-cold PBS and then homogenized in extraction buffer, as described by the manufacturer. After incubation on ice, the extract was centrifuged at 10,000 × g at 4°C for 5 min, and 50 μl of supernatant was mixed with an equal volume of 2 × reaction buffer and 2 μl of substrate in a 96-well microplate. Plates were kept in the dark at 37°C for 1 h, and fluorescence was recorded using a microplate reader (Bio-Rad, CA, United States). Protein concentration was determined by the Bradford assay (Bio-Rad, CA, United States). Cathepsin B, D, and K activities were determined by fluorometry at Ex/Em = 400/505, 328/460, and 400/505 nm, respectively.

Enzyme activity assays were performed on the three available AVM-derived primary cell lines used for western blot analysis using enzyme activity assay kits to determine enzymatic activity of cathepsin B (cat#ab65300, Abcam), and cathepsin D (cat#ab65302, Abcam). Assay procedure was carried out according to manufacturer protocol. Due to a very small dynamic range for the cathepsin D activity assay, a titration was carried out using a recombinant cathepsin D protein to validate the assay. A further titration assay was carried out using tissue and cell line samples to establish the appropriate amount of protein to add for a valid assay result. Fluorescence was measured in a black 96-well plate (cat#3631, Corning, ME, USA) using the Varioskan Flash plate reader (cat#MIB5250030, Thermo Fisher Scientific). Human tonsil tissue and HepG2 cell lysates were used as appropriate positive and denatured for use as negative controls.

BMDCs (2 × 10⁶) were cultured in 12-well plate with phenol red-free RPMI complete medium (Invitrogen) and stimulated with or without H. capsulatum at MOI of 20. Cells and culture supernatants were collected separately 18 h after stimulation. Cell-free supernatants were concentrated 10-fold by Vivaspin 500 (GE Healthcare). Cells were lysed with cathepsin B lysis buffer (Abcam, ab65300). Both cell lysates and concentrated supernatants were loaded unto 96-well plate (solid black, Corning) before addition of CB reaction buffer. CB substrate Ac-RR-AFC (final concentration of 200 μM) was added and incubated at 37°C for 1 h. Samples were read in SpectraMax M5 (Molecular Devices) at 400 nm excitation and 505 nm emission wavelengths.