Cardiac CatB activity was determined by using a commercial Cathepsin B Activity Assay Kit (Fluorometric) (Abcam, England) according to the instructions. The appropriate amount of heart tissues lysates was incubated with CatB substrate in the 96-well black plates with clear bottoms for 60–120 minutes at 37°C protected from light. The fluorometric absorbance was then measured at the Ex/Em of 400/505nm by using the microplate reader (Bio-Rad, Hercules, CA). The results were shown as relative fluorescence units (RFU) per microgram of protein.
Cathepsin b activity fluorometric assay kit
Manufactured by Abcam
Sourced in United Kingdom, United States
The Cathepsin B Activity Fluorometric Assay Kit is a laboratory tool used to measure the activity of the enzyme Cathepsin B. Cathepsin B is a cysteine protease involved in various cellular processes. The kit uses a fluorogenic substrate to quantify Cathepsin B activity in biological samples.
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Lab products found in correlation
Cell lysis buffer, by Abcam (2 mentions)
Microplate reader, by Bio-Rad (1 mentions)
Infinite 200 pro, by Tecan (1 mentions)
Nanodrop onec uv vis spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Infinite m200 pro multimode microplate reader, by Tecan (1 mentions)
P62 ab56416, by Abcam (1 mentions)
Lamp2a ab18528, by Abcam (1 mentions)
Anti nbr1, by Cell Signaling Technology (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Chloroquine diphosphate salt, by Merck Group (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Fluostar optima plate reader, by BMG Labtech (1 mentions)
Vectashield hardfset mounting medium with dapi, by Vector Laboratories (1 mentions)
Ecl prime western blotting detection reagent, by GE Healthcare (1 mentions)
Vectastain abc kit, by Vector Laboratories (1 mentions)
12 protocols using cathepsin b activity fluorometric assay kit
1
Cathepsin B Activity Assay in Cardiac Tissue
2
Cathepsin B Activity Assay in LLC Tumors
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Mice with subcutaneous LLC tumors, which were treated with the vehicle, ACC, and cisplatin, were resected after the study’s termination and animal euthanasia (n = 8, per group). All conditions of maintenance and handling were similar between the groups.
For the assay, 20 mg of each extracted tumor was weighed and lysed. Measurements were performed using the Cathepsin B Activity Assay Kit (Fluorometric) (ab65300 by Abcam, Cambridge, UK). The assays were performed on tumors from three groups, excluding the group that received the combined cisplatin and ACC treatment due to a low tumor weight. The lysed samples were treated per the kit’s instructions. In brief, 50 μL of lysis buffer was used, and the cells were incubated on ice for 10 min. Centrifugation was carried out at 20,000× g for 5 min, and then the supernatant was transferred to a new tube. In total, 50 μL of the lysate was added to an opaque black 96-well plate. Then, 50 μL of the reaction buffer and 2 μL of the 10 mM substrate Ac-RR-AFC were added to each sample. In this study, 2 μL of the inhibitor was used as the negative control. The Infinite®200Pro (Tecan, Männedorf, Switzerland) microplate reader was used with 400 nm excitation and a 505 nm emission filter to analyze the fluorescence intensity for cathepsin B enzymatic activities after incubating the samples at 37 °C for 1 h in the dark.
For the assay, 20 mg of each extracted tumor was weighed and lysed. Measurements were performed using the Cathepsin B Activity Assay Kit (Fluorometric) (ab65300 by Abcam, Cambridge, UK). The assays were performed on tumors from three groups, excluding the group that received the combined cisplatin and ACC treatment due to a low tumor weight. The lysed samples were treated per the kit’s instructions. In brief, 50 μL of lysis buffer was used, and the cells were incubated on ice for 10 min. Centrifugation was carried out at 20,000× g for 5 min, and then the supernatant was transferred to a new tube. In total, 50 μL of the lysate was added to an opaque black 96-well plate. Then, 50 μL of the reaction buffer and 2 μL of the 10 mM substrate Ac-RR-AFC were added to each sample. In this study, 2 μL of the inhibitor was used as the negative control. The Infinite®200Pro (Tecan, Männedorf, Switzerland) microplate reader was used with 400 nm excitation and a 505 nm emission filter to analyze the fluorescence intensity for cathepsin B enzymatic activities after incubating the samples at 37 °C for 1 h in the dark.
3
Cathepsin B Activity Assay in BMDCs
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Cathepsin B activity was measured with Cathepsin B Activity Assay Kit (Fluorometric) (abcam, Cambridge, UK). BMDCs (1.0×10 6 cells) were treated with each carrier at siRNA concentrations of 10, 30 and 100 nM for 2 h at 37°C in 0.5 mL of serum-free OPTI-MEM I in microtube. After the incubation, the cells were collected by centrifugation and suspended in 0.5 mL of ice-cold PBS. Digitonin solution was then added to the cell suspension. The permeabilization of only the plasma membrane was performed by treatment with a 32 µg/mL digitonin solution, whereas that of cell membrane and endosomal membrane was performed using a 200 µg/mL digitonin solution. After incubation for 15 min on ice, the supernatant was collected by centrifugation. 50 µL of the supernatant was mixed with 50 µL of cathepsin B substrate reagent in 96 well plate, followed by incubation for 2 h at 37°C. Finally, FI was measured by EnSpire 2300 Multilabel Reader with λex = 400 nm, λem = 505 nm. The FI of no treatment cells treated with 32 µg/mL digitonin was set to 0% of cathepsin B activity, whereas the FI of the cells treated with 200 µg/mL digitonin was set to 100% of cathepsin B activity.
4
Cathepsin B Activity in Hypoxic LNCaP Cells
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LNCaP cells were seeded in wells of 24 well-plates at a concentration of 6 × 104 cells per well and incubated for 24 h under normoxic conditions. The growth medium was then removed, and the cells were incubated in a hypoxic (2 mmHg O2) environment, at pH 6.4 or pH 7.4, for 24 h. Intracellular and extracellular cathepsin B activity was determined using a fluorometric cathepsin B activity assay kit (Abcam, UK), from lysed cells and the corresponding growth medium, respectively. The incubation medium was collected for extracellular cathepsin B analysis. For the intracellular cathepsin B assessment, cells were lysed using a cell lysis buffer (Abcam, UK) according to the manufacturer's instructions. Subsequently, 50 μL aliquots of each cell lysate or extracellular medium sample were mixed with 50 μL of cathepsin B reaction buffer and 2 μL of cathepsin B substrate. Samples were incubated for 45 min and fluorescence was measured (Ex/Em = 400/505 nm). The data were normalised using the total protein concentration (mg/mL) in the cell lysate or extracellular medium samples.
5
Cathepsin B Activity in Hypoxic Tumor Cells
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BxPC-3, PANC-1, and T110299 cells were seeded in 24-well plates at a concentration of 6 × 104 cells per well and incubated for 24 h, in a humidified 5% CO2/20% O2 atmosphere, at 37 °C. The growth medium was then removed, and the systems incubated at either normoxic (20% O2) conditions and pH 7.4 or at hypoxic (1% O2) conditions and pH 6.4, for 24 h. A fluorometric cathepsin B activity assay kit (Abcam) was used to determine intracellular and extracellular cathepsin B activity from lysed cells and the corresponding growth medium recovered from the culture wells, respectively. The growth medium was recovered for extracellular cathepsin B analysis. For intracellular activity, cells were lysed using cell lysis buffer, (Abcam) based on the manufacturer’s instructions. The lysate and extracellular medium samples were both normalised based on total protein concentration (mg/mL), measured using a NanoDrop OneC UV–Vis spectrophotometer (ThermoFisher Scientific). Subsequently in the wells of a 96-well plate, 50 µL of lysate/extracellular medium sample, 50 μL of cathepsin B reaction buffer and 2 µL of cathepsin B substrate were added. The plates were then incubated for 45 min, and fluorescence was measured using an Infinite M200 Pro Multimode Microplate Reader (Tecan, Switzerland) at 400 nm and 505 nm excitation and emission wavelengths, respectively (Ex/Em = 400/505 nm).
6
Autophagy-Related Protein Detection
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The following antibodies were purchased from Abcam: p62 (ab56416), LAMP2A (ab18528), SNAP29 (ab138500), VAMP8 (ab75021). The STX17 antibody was purchased from Proteintech (17815-1-AP). The antibody against LC3B (for western blots and immunofluorescence (IF) with p62) was purchased from Novus Biologics (NB100-2220). The secondary goat anti-rabbit IgG antibody was purchased from R&D systems (HAF008). The Alexa Fluor® 488 secondary goat anti-mouse IgG antibody was purchased from Invitrogen (A11001). The following were purchased from Sigma-Aldrich: Chloroquine diphosphate salt (C6628), and anti-β-actin (A5441). The following antibody was purchased from Cell Signaling: anti-NBR1 (#9891). The following were purchased from Jackson ImmunoResearch: Rhodamine RedTM goat anti-mouse IgG secondary antibody for TRITC (115-295-146), Alexa Fluor® 488 goat anti-rabbit IgG secondary antibody for FITC (111-545-144), HRP goat anti-mouse secondary antibody (115-036-003). The Lysotraker Red DND-99 reagent was purchased from Invitrogen (L752). Cathepsin B activity fluorometric assay kit was purchased from Biovision (#K-140).
7
Measuring Cathepsin B Activity in Cellular Fractions
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Cathepsin B activity was measured using the Cathepsin B Activity Fluorometric Assay Kit (BioVision; Milpitas, CA, USA). Lysosomal and cytosolic fractions were isolated as described above. Lysosomal membranes were disrupted by three freeze thaw cycles and protein concentration of the fractions was determined by Bradford assay (Bio-Rad; Hercules, CA, USA). Equal amounts of sample and reaction buffer (50 μL) were added to a 96-well plate and incubated with 2 μL of cathepsin B substrate sequence RR labeled with amino-4-trifluoromethyl coumarin (Ac-RR-AFC) for 2 h at 37°C. A sample with cathepsin B inhibitor was run in parallel. Fluorescence (excitation 400 nm/emission 505 nm) was measured on a FLUOstar OPTIMA plate reader (BMG Labtech; Ortenberg, Germany) spectrofluorometer with FLUOstar OPTIMA software version 1.32R2. Fold-increase in cathepsin B activity was determined by comparing the relative fluorescence units/mg protein after subtracting the sample with the inhibitor.
8
Measuring Cathepsin B Proteolytic Activity
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To measure the proteolytic activity of cathepsin B (CTSB), we used the Cathepsin B Activity Fluorometric Assay Kit (Biovision) according to the manufacturer's instructions.
9
Cathepsin B Activity Assay
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Purified human cathepsin B (Calbiochem) was activated by incubation at 37°C with 5 mM DTT in 50 mM Tris (pH 6.1). The activity of cathepsin B was then measured in presence or absence of 10 μM Ca074 using the Cathepsin B Activity Fluorometric Assay Kit from BioVision according to the manufacturer's instructions (BioVision, Milpitas, CA). Subconfluent HT29 and HEK293 cells were incubated with 10 μM Ca074 for 12 h. Cells were then washed twice with PBS, lysed in cell lysis buffer provided in the assay kit and the resulting activity measured according to the manufacturer's instructions.
10
Cathepsin B Activity Quantification
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After SEA treatment for 24 h, cells were washed twice with PBS, scraped in lysis buffer to extract proteins, and measured cathepsin B activity in HSCs by using a commercially available cathepsin B activity fluorometric assay kit (Biovision) according to the protocol described by the manufacturer.
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