Following drug treatment, cells were incubated with 0.5 μM LysoTracker Red DND-99 (Life Technologies, L7528) or 0.75 μM MitoTracker Red CMXRos (Life Technologies, M7512) for 30 min at 37 °C. Subsequently, cells were washed twice with PBS. Fluorescent images of live cells were observed under an Axio Observer A1 microscope (Carl Zeiss). Cathepsin B activity was examined using the Magic Red Cathepsin B detection kit (ImmunoChemistry Technologies, 937). Briefly, Magic Red Cathepsin B reagent was added to the cell medium, and after 1 h, cells were washed twice with PBS. Fluorescent images were taken using an Axio Observer A1 microscope. For monitoring autophagic flux, MN9D cells plated on poly-D-lysine-coated culture dishes were cultivated for 2 days and subjected to transient transfection with an mRFP-EGFP tandem fluorescent-tagged LC3 probe (a generous gift from Prof. Tamotsu Yoshimori at Osaka University, Japan) for 24 h using Lipofectamine 2000 (Thermo Fisher Scientific, 11668019) as recommended by the supplier. Drug treatment was performed 24 h post-transfection.
Magic red cathepsin b detection kit
Manufactured by ImmunoChemistry Technologies
Sourced in United States
The Magic Red Cathepsin B Detection Kit is a fluorescence-based assay designed to detect and quantify the activity of the lysosomal protease Cathepsin B in cells and tissues. The kit employs a specific substrate that is cleaved by Cathepsin B, resulting in the release of a red-fluorescent dye that can be detected using a fluorescence microscope or plate reader.
Automatically generated - may contain errors
Lab products found in correlation
Axio observer a1 microscope, by Zeiss (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Mitotracker red cmxros, by Thermo Fisher Scientific (1 mentions)
Lysotracker red dnd 99, by Thermo Fisher Scientific (1 mentions)
Te2000 u, by Nikon (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Lysotracker deep red, by Thermo Fisher Scientific (1 mentions)
Sensolyte 520 cathepsin b assay kit, by AnaSpec (1 mentions)
4 protocols using magic red cathepsin b detection kit
1
Monitoring Lysosomal, Mitochondrial, and Autophagic Activities
2
Lysosomal Function Monitoring in H2O2-Treated Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
LysoTracker Deep Red is an ideal fluorescent acidotropic probe that selectively labels vacuoles with low internal pH. Thus, it can be used to label and track functional lysosomes. Briefly, the cells were treated with H2O2 in the presence or absence of NaHS and were then incubated with the LysoTracker Deep Red (70 nM, Life Technologies) in a pre-warmed medium at 37°C for 30 min. Subsequently, the solution was replaced with fresh medium, and the cells were observed using a fluorescence microscope (TE-2000U, Nikon). The activity and intracellular distribution of cathepsin B, a predominant lysosomal protease, was assessed with Magic Red Cathepsin B Detection Kit (Immunochemistry Technologies, LLC, Bloomington, MN, USA). The cells were stained with MagicRed Cathepsin B substrate for 30 min at 37°C and then washed twice with phosphate buffered saline (PBS). Finally, the cells were stained with DAPI (1 μg/ml, Sigma-Aldrich) for 10 min and observed with a fluorescence microscope (TE-2000U, Nikon).
3
Measuring Cathepsin B Activity in Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cathepsin B activity was measured using the Magic Red Cathepsin B Detection Kit (ImmunoChemistry Technologies, 937) as previously described12 (link). Briefly, control or APAP-treated HEI-OC1 cells were cultured in confocal dishes (for microscopy) for different lengths of times, as indicated. The cells were then loaded with Magic Red Cathepsin B reagent for 1 h, followed by washing twice with PBS. More than ten fluorescence images were taken using a confocal microscope and representative images are shown. ImageJ software was used to quantify the cathepsin B activity.
4
In Situ and Extracellular Cathepsin B Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To detect cathepsin B enzymatic activity in situ, Cbln1-SEP signals were imaged, and then red-channel signals were taken after granule cells were treated with MagicRed solution (1:20 dilution) for 12 min (MagicRed Cathepsin B detection kit, #937, ImmunoChemistry Technologies) and washed.
To detect cathepsin B enzymatic activities in the culture medium, cleavage of QXL520/HiLyte Fluor488 peptide substrate (SensoLyte 520 Cathepsin B Assay Kit, AS72164, AnaSpec) was measured using fluorescence resonance energy transfer according to the manufacturer's protocol.
To detect cathepsin B enzymatic activities in the culture medium, cleavage of QXL520/HiLyte Fluor488 peptide substrate (SensoLyte 520 Cathepsin B Assay Kit, AS72164, AnaSpec) was measured using fluorescence resonance energy transfer according to the manufacturer's protocol.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!