Lysosomal proteolytic function was examined using either DQ-red-BSA (Thermo Fisher Scientific) or the Magic Red cathepsin B kit (Bio-Rad). Briefly, cells were washed after NDS treatment, then incubated with a substrate (10 μg/mL DQ-red-BSA or 1:1000 dilution Magic Red) at 37°C for 1 h. After removing the medium, the cells were washed twice with PBS and imaged using confocal fluorescence microscopy. Image analysis was performed with Image J (NIH, Bethesda, MD). The integrated fluorescence intensity from the maximum projection of each image stack was used to compare substrate degradation rates.
Magic red cathepsin b kit
Manufactured by Bio-Rad
The Magic Red Cathepsin B kit is a fluorescence-based detection system designed to measure the activity of the lysosomal protease Cathepsin B in live cells. It utilizes a fluorogenic substrate that becomes fluorescent upon cleavage by Cathepsin B, allowing for the quantification of its enzymatic activity.
Automatically generated - may contain errors
Lab products found in correlation
Spinning disk confocal head, by Oxford Instruments (2 mentions)
Dq red bsa, by Thermo Fisher Scientific (1 mentions)
Lysosensor green dnd 189, by Thermo Fisher Scientific (1 mentions)
Metamorph software, by Molecular Devices (1 mentions)
Thunder imager 3d cell culture microscope, by Leica (1 mentions)
5 protocols using magic red cathepsin b kit
1
Lysosomal Proteolytic Function Assay
2
Lysosomal Imaging and Cathepsin Activity
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Cells on coverslips were loaded for 60 min in a 5% CO2 atmosphere at 37°C with 1 μM LysoSensor Green DND-189 (Invitrogen) in culture medium before being mounted onto a temperature-controlled microscope stage (QE-2 Quick Exchange Platform; Warner Instruments). Imaging was performed at 37°C in physiological buffer (145 mM NaCl2, 5 mM NaHCO3, 2 mM KCl, 2.5 mM MgCl2, 1 mM CaCl2, 10 mM HEPES, 20 mM glucose, pH 7.4/NaOH) using MetaMorph software (Molecular Devices). Cathepsin activity was imaged in live cells in physiological buffer at 37°C using the Magic Red Cathepsin B kit (Bio-Rad Laboratories) according to the manufacturer’s protocol.
3
Quantifying Cathepsin B Activity in RPTECs
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The Magic Red Cathepsin B Kit (#ICT938; Bio-Rad) was used to quantify and monitor cathepsin B activity in RPTEC/TERT1 cell lines. Briefly, differentiated control, CLCN5 KD, and ClC-5 rWT RPTECs were treated with either DMSO (vehicle) or bafilomycin A1, as a positive control, for 24 h. Next day, cells were washed three times with isotonic solution containing 2.5 mM KCl, 140 mM NaCl, 1.2 mM CaCl2, 0.5 mM MgCl2, 5 mM glucose, and 10 mM Hepes (305 mosmol/litre, pH 7.4 adjusted with Tris), and incubated with Magic Red cathepsin B reactive at 1:26 (vol/vol) for 5 min at 37°C (following the manufacturer’s protocol). After the incubation period, the cells were imaged using Leica Thunder Imager 3D Cell Culture Microscope. All data were processed and analysed using FIJI (47 (link)).
4
Quantifying Lysosomal Cathepsin B Activity
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The Magic Red Cathepsin B kit (Bio-Rad, ICT937) was used to analyze the protease activity of Cathepsin B in lysosomes as a proxy to lysosome function. In the presence of functional cathepsin B, the Magic Red substrate is cleaved allowing the Cresyl violet fluorophore to fluoresce red upon excitation at 550-590 nm. Briefly, cells to be analyzed were plated on coverslips and the Magic Red substrate (Magic Red stock was reconstituted in 50 μl DMSO and diluted 1:10 in deionized water) was added to the growth media (20μl was added per 300μl of growth media as per the manufacturer’s instructions) for the final hour of the experiment. Cells were then fixed in 4% formaldehyde as previously described. Cresyl Violet fluorescence was detected using an inverted Nikon microscope coupled with a spinning disk confocal head (Andor). Z stack images were acquired, and sum intensity image projections were generated using ImageJ. Fluorescence intensity was then quantified per cell with ImageJ66 (link). All conditions were quantified blindly.
5
Lysosomal Cathepsin B Activity Assay
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The Magic Red™ Cathepsin B kit (Bio-Rad, ICT937) was used to analyze the protease activity of Cathepsin B in lysosomes as a proxy to lysosome function. In the presence of functional cathepsin B, the Magic Red substrate is cleaved allowing the Cresyl violet fluorophore to fluoresce red upon excitation at 550-590 nm. Briefly, cells to be analyzed were plated on coverslips and the Magic Red substrate (Magic Red stock was reconstituted in 50 µl DMSO and diluted 1:10 in deionized water) was added to the growth media (20µl was added per 300µl of growth media as per the manufacturer's instructions) for the final hour of the experiment. Cells were then fixed in 4% Formaldehyde as previously described. Cresyl Violet fluorescence was detected using an inverted Nikon microscope coupled with a spinning disk confocal head (Andor). Z-stack images were acquired, and sum intensity image projections were generated using Image J. Fluorescence intensity was then quantified per cell with ImageJ [61] . All conditions were quantified blindly.
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