Name: Magic Red Cathepsin B Assay
SKU: 937, 938
Availability: InStock

Elevated cathepsin enzyme activity in serum or the extracellular matrix can signify a number of pathological conditions. ICT’s Magic Red assay kits are used by researchers seeking to quantitate and monitor cathepsin activity in cultured cells and tissues. This assay kit detects cathepsin B activity.

To use Magic Red, add the substrate directly to the cell culture media, incubate, and analyze. Because it is cell permeant, it easily penetrates the cell membrane and the membranes of the internal cellular organelles – no lysis or permeabilization steps are required. If cathepsin enzymes are active, the Magic Red substrate is cleaved and the cresyl violet fluorophore will become fluorescent upon excitation. As enzyme activity progresses and more Magic Red substrate is cleaved, the signal will intensify, allowing researchers to watch the color develop over time. Samples can be analyzed by fluorescence microscopy or with a fluorescence plate reader. Hoechst 33342 and Acridine Orange are included in the kit to detect nuclear morphology and lysosomal organelle structure, respectively.

1. Prepare samples and controls.
2. Reconstitute Magic Red by adding 50 µL DMSO.
3. Dilute Magic Red 1:10 by adding 450 µL diH₂O.
4. Add 20 µL Magic Red to each sample (480 µL aliquot of cultured cells).
5. Incubate while protected from light.
6. Watch color start to develop within 15 minutes.
7. If desired, label with additional stains, such as Hoechst 33342, DAPI, or an antibody.
8. If desired, fix cells.
9. Analyze with a fluorescence microscope or fluorescence plate reader. Magic Red has an optimal excitation at 592 nm and emission at 628 nm.

Cathepsin B-positive THP-1 cells fluoresce red after staining with MR-(RR)2. Red fluorophore concentrates inside lysosomes (Dr. Brian W. Lee, ICT).

• Reagent name: MR-RR₂
• Target: Cathepsin-B
• Excitation/Emission: 592 nm / 628 nm
• Method of Analysis: Fluorescence Plate Reader, Fluorescence Microscope
• Types of Samples: Cell culture
• Storage: 2-8°C
• Shipping: Ships overnight (domestic), International Priority Shipping

Kit 937: 25 Tests
• Magic Red Substrate (MR-RR₂), 1 25 Test Vial, #6133
• Hoechst 33342, 1 mL, #639
• Acridine Orange, 0.5 mL, #6130
• Kit Manual

Kit 938: 100 Tests
• Magic Red Substrate (MR-RR₂), 1 100 Test Vial, #6134
• Hoechst 33342, 1 mL, #639
• Acridine Orange, 0.5 mL, #6130
• Kit Manual

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Question: When you dilute the stock concentration 1/10 in H2O and aliquot is for future use, how long can this be stored? I see that the stock concentration in DMSO can be stored for 6 months.

Answer: Once the stock solution is diluted 1:10 in diH2O we recommend using within 15 minutes to prevent hydrolysis of the Magic Red substrate. Non-specific hydrolysis would lead to fluorescence not associated with enzymatic cleavage of the substrate. We do not recommend attempting to aliquot or store the diH2O-diluted substrate solution for future use.

Yes, that’s correct. The DMSO-reconstituted Magic Red stock can be stored frozen for up to 6 months protected from light and thawed more than twice during that time.