Quantitate and monitor intracellular cathepsin-B activity over time in vitro. The Magic Red substrate in this assay fluoresces red upon cleavage by active cathepsin enzymes. Analyze the fluorescent signal using fluorescence microscopy or a fluorescence plate reader.
- Prepare samples and controls
- Reconstitute Magic Red by adding DMSO.
- Dilute Magic Red 1:10 by adding diH2O.
- Add 20 µL Magic Red to each sample (~ 500 µL aliquot of cultured cells).
- Incubate while protected from light.
- Watch color start to develop within 15 minutes.
- If desired, label with additional stains, such as Hoechst, DAPI, or an antibody.
- If desired, fix cells.
- Analyze with a fluorescence microscope or fluorescence plate reader. Magic Red has an optimal excitation at 592 nm and emission at 628 nm.
If working with adherent cells, please see the manual for additional protocols.
Product Specific References
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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.