CATHEPSIN B K L APOPTOSIS CASPASE DETECTION KITS FOR IN VITRO USE IN REAL TIME MAGIC RED

Magic Red™ Real-Time Cathepsin Detection Kits
Visualize cathepsin activity in whole living cells.

Cathepsin B 
Cathepsin K 
Cathepsin L

Rat fibroblasts were seeded in 12 well plates at 10,000 cells in 1mL and irradiated the following day. 500uL was removed; 23.1uL of MR-DEVD solution (cat. #935) was mixed with 200uL media and added. 1 hour later, 300uL media was added and cells were photographed over 16 hours. Bright field image shows increasing red intensity as caspase activity and apoptosis progressed. Time 0 is at left, 16 hours is at right.  Data courtesy of Dr. Martin Purschke, MA General Hospital. Watch the color increase in the movie clip of these cells in a bright field.     

top = apoptotic
bottom = caspase-negative

ICT's MR Caspases 3&7 kit clearly distinguishes apoptotic from non-apoptotic cells. The top 3 THP-1 cells have active caspases (they fluoresce red) the bottom 3 cells do not (they do not fluoresce). Read this publication

     ICT's Magic Red™ kits enable you to monitor cathepsin activity over time and will give you a clear picture of cathepsin-positive versus cathepsin-negative cells. Just add MR to the media and analyze. As protease activity increases, positive cells will start to fluoresce.
     These unique kits are not ELISAs and do not use antibodies - instead they use cell-permeant substrates specifically targeted by active enzymes. The target substrate peptide sequence is linked to a red fluorophore, Magic Red™ (also known as cresyl violet), which fluoresces once the substrate is cleaved by the specific enzyme (cathepsin B, K, or L). As cathepsin activity progresses and more substrate is cleaved, the red fluorescent signal increases. Only cells with active enzymes will fluoresce, so you don’t get any signal from pro-enzymes or inactive forms of the enzyme. ICT’s MR kits give you a clear picture of protease-positive versus protease-negative cells. MR kits are true substrate assays - the red fluorescent label is quenched until it is actually cleaved by the active enzyme, so you can actually see the color develop over time. Watch 2 movies of real-time protease activity, courtesy of Dr. M. Purschke, MA General Hospital: movie clip of live cells (bright field) ; movie of red fluorescence only. 
     Just culture your cells, add MR to the media, incubate, and analyze. Once MR is added to the media, it will pass through the cell membrane - no lysis or permeabilization steps are required. If it is cleaved by an active cathepsin, the MR fluorophore will stay inside the cell, and often aggregates inside lysosomes. Cathepsin enzymes are lysosomal. Watch the cells through a microscope (and protect from light when not taking pictures) or read total fluorescence of the sample with a fluorescence plate reader (using a black microtiter plate).
     ICT's Magic Red™ kits do not need a wash step. In fact, we discourage additional manipulation of the cells as the MR fluorophore may migrate back out of the cell. Adherent cells can be trypsinized and transferred into a black microtiter plate for analysis with a fluorescence plate reader. We have successfully used these kits with Jurkat, HL-60, THP-1, fibroblasts, UMUC-3, MCF-7, and U937 cells. Protect the cells from light as the reagent will photobleach over time. 
     MR kits work well with cells that cannot be centrifuged, with adherent cells, with thin frozen sections, and can be adapted for hi-throughput screening.  You can use FLICA™ and MR together for dual-staining studies.  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. 

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sensitive.

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Dual staining of apoptotic MCF-7 cells reveals areas of general caspase activity, indicated by green fluorescence from FAM-FLICA™ Poly-Caspases Kit (cat. # 92). Red fluorescence generated from MR-Caspases 3&7 (cat. # 936) indicates areas of caspase 3 activity (MCF-7 cells are deficient in caspase 7).

Frequently Asked Questions

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.

References
Lee, B., G. L. Johnson, S. A. Hed, Z. Darzynkiewicz, J. Talhouk, and S. Mehrotra.  2001.  DEVDase detection in intact apoptotic cells using the cell permeant fluorogenic substrate, (z-DEVD)₂-cresyl violet.  Biotechniques vol 35 no 5:1080-1085. Read this publication .

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Cathepsin B

Cathepsin B-positive THP-1 cells fluoresce red after staining with MR-RR. Red fluorophore concentrates inside lysosomes.

our Cathepsin B page

MR-Cathepsins Manual

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Cathepsin K

Cathepsin K-positive Jurkat cells fluoresce red after staining with MR-LR.

     ICT's Magic Red™-LR kits measure active cathepsin K in whole living, intact cells - no lysis required. As cathepsin K activity increases, the red fluorescent signal increases. Read more on our Cathepsin K page

MR-Cathepsins Manual

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Cathepsin L

Cathepsin L-positive HL-60 cells fluoresce red after staining with MR-FR.

      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. Read more on our Cathepsin L page

MR-Cathepsins Manual

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Frequently Asked Questions about Magic Red™

How sensitive is it? MR kits are very sensitive. Protease-positive cells often generate a signal 2-5x greater than the negative cell population when read in fluorescence plate readers.

How specific is it? MR kits are very specific. However, there still can be some cross-reactivity as the reagent is incubating (often for several hours). The sequence used for the MR Caspase Apoptosis kit, DEVD, will be cleaved by both caspase 3 and caspase 7. We designed MR using the most specific substrate sequences: Cathepsin B uses RR; Cathepsin K uses LR; and Cathepsin L uses FR. ICT's Cathepsin B (MR-RR) reagent is the most specific of the cathepsin reagents - there is very little cross reactivity with other proteases. Cathepsin B is highly abundant and highly active. The Cathepsin K and L reagents have a preference for the target sequences, MR-LR and MR-FR respectively. Although they have their preferred amino acid sequence motifs, they are still capable of reacting with other less desirable substrate sequences albeit at a lower efficiency in terms of kcat/Km ratios. 

How long does the assay take? The MR substrate will start to react with proteases as soon as it is added to the media. Color may be visualized in positive cells within 15 minutes of addition to the cells. Typical incubation periods are 1-4 hours; we have incubated cells with MR for 72 hours without killing the cells. You can start and finish your experiment in one day. You may even be able to finish in 1-2 hours, depending upon the reactivity of your particular cell line. Adherent cells can be trypsinized prior to analysis in a plate reader. The exact incubation time should be determined by your experimental protocol.  

What is “1 test”? To standardize the manual, “1 test” is a 300uL aliquot of suspension cells grown up to 1x10^6 (maximum) for testing on a fluorescence plate reader. We typically analyze 200,000 cells/well in a plate reader. MR also works with just a few cells for viewing under a fluorescence microscope.

How much reagent do I use? MR reagents are provided as stabilized lyophilized powders. The vial may appear empty, but it's not. The reagent will appear as an iridescent sheen inside the vial. Reconstitute the small "25 test" vial with 50uL DMSO; reconstitute the large "100 test" vial with 200uL DMSO. Dilute 1:5 with PBS to create the 31X working stock. Add ~10uL of the 31X working stock to your cells. Your particular cells may require more or less reagent. The first time you use the kit, set up a titration experiment to determine the optimal amount of reagent to add to your cells. Protect the reagent and treated cells from light as the reagent will photobleach over time. 

What wavelengths do I need? MR reagents excite at 540-590 and emit at >610nm. Other filter pairings can be used which closely approximate these ranges. Analyze with a fluorescence microscope, a fluorescence plate reader, or special flow cytometers with adjustable excitation wavelengths (call us for FACS details).  

Can I use it in combination with other fluorescent reagents?  Yes. MR kits include Hoechst (blue; ex. 365, em. 480) and acridine orange (orange; ex. 550, em. 610), so you can do dual-staining studies for DNA and lysosomes. You can also use MR with ICT's FAM-FLICA™ and FAM- FLISP™ kits (green; ex. 490, em. 520). LysoTracker Green (ex. 504, em. 511) from Molecular Probes seems to be a good fit with MR, it is well below the optimal excitation and emission ranges, but has an extremely short Stokes Shift of only 7 nm, meaning that it is essentially read right on top of the excitation line (ICT has not tested LysoTracker Green).

What types of cells have you tried? Jurkat, HL-60, U937, THP-1, MCF-7, UMUC-3, fibroblasts.

Will it work with fixed or paraffin or frozen cells? Magic Red™ kits will not work on fixed cells (but you can fix the cells after labeling), nor will they work with paraffin embedded cells. The reagents require the enzymes to be active, and fixation and paraffin inactivates the enzymes. Magic Red™ kits may work with some frozen cells and thin frozen sections. The reagents require the cell membrane to remain intact to contain the red fluorophore within the cell. Freezing often creates pores in the membranes, allowing the positive fluorescent signal to leave the cell. If this is a problem, all the cells will appear red as the fluorophore travels throughout the sample. Magic Red™ kits may work best with frozen cells when measuring total fluorescence with a plate reader.

How do I wash the cells? Magic Red™ MR kits do not require a wash step. We discourage additional manipulation of the cells as the MR fluorophore may migrate back out of the cell.

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