Magic Red™ Cathepsin L Detection Kit
Assess cathepsin L activity in vitro and in real time
ICT's Magic Red™-FR kits measure active cathepsin L in whole living, intact cells - no lysis required. As cathepsin L activity increases, the red fluorescent signal increases. These unique kits are not ELISAs and do not use antibodies - instead they use a cell-permeant substrate sequence, FR, specifically targeted by active cathepsin L. This peptide sequence is linked to a red fluorophore, Magic Red™, which fluoresces once cleaved by active cathepsin L. Only cells with active cathepsin L will fluoresce, so you don’t get any signal from pro-enzymes or inactive forms of the enzyme. ICT’s MR-FR kits give you a clear picture of cathepsin-L-positive versus negative cells.
Cathepsin-L-positive cells often generate a signal 2-5x greater than the negative cell population when read in fluorescence plate readers. MR excites at 540-590 and emits at >610nm. This signal can be detected with a fluorescence microscope, a fluorescence plate reader, or special flow cytometers with adjustable excitation wavelengths.
MR kits are very specific. However, there still can be some cross-reactivity as the reagent is incubating (often for several hours). The Cathepsin L reagent has a high preference for the target sequence, MR-FR. Although Cathepsin L has its preferred amino acid sequence motif, it is still capable of reacting with other less desirable substrate sequences albeit at a lower efficiency in terms of kcat/Km ratios.
In these cells we are seeing the hydrolysis of the MR-FR substrate, which has a preference for Cathepsin L. However, the reagent could by cleaved by other less desirable proteases albeit at a lower efficiency in terms of kcat/Km ratios. Cathepsin L is lysosomal. After staining with MR-FR, there are often areas of bright red fluorescence as the reagent is processed and concentrated within the lysosomal bodies.
Some characteristics of cathepsin L:
1. Responsible for normal protein breakdown activity within the lysosome.
2. Associated with disease states such as excessive bone resorption, tumor metastasis, and rheumatoid arthritis.
3. An abundant lysosomal protease along with cathepsin B and D.
4. Requires an acidic pH environment such as that found within lysosomes for enzyme activity.
5. An endopeptidase.
Magic Red Cathepsin L Detection kit contains:
- ICT's red Magic Red cathepsin L detection probe, MR-(FR)2
- Hoechst stain (a blue DNA stain)
- Acridine orange (a lysosomal stain)
- Acridine orange (a lysosomal stain)
- MR-Cathepsins Manual
Trial size, ~25 tests:
Regular size, ~100 tests:
Cathepsin L-positive HL-60 cells fluoresce red after staining with MR-FR.
Sample Protocol
1. Culture your cells individually or up to 1 x 106 cells/mL.
2. Induce your experimental conditions following your protocol.
3. Reconstitute the reagent with DMSO to form the stock concentrate (which can be frozen for future use).
4. Dilute the stock concentrate with 1X PBS to form the working solution.
5. Add ~10uL of the working solution directly to a 300-500uL aliquot of your cell culture for labeling.
6. Incubate, typically 1-4 hours; protect cells from light.
7. If desired, label DNA with Hoechst stain.
8. If desired, label lysosomes with Acridine Orange.
9. If desired, fix cells.
10. Analyze data using a fluorescence microscope, or plate reader.