Magic Red™ Cathepsin K Assay Kit

Catalog Number: 939 (25 tests), 940 (100 tests)

Availability: In stock

$164.00

Quick Overview

Magic Red™ Cathepsin K detection kits provide a simple method to monitor the varying constitutive levels of generalized cathepsin K enzyme activity in whole, living cells.
Our Magic Red™ Cathepsin K assay is based on the cresyl violet (CV) fluorogenic substrate CV-(Leu-Arg)2 or CV-(LR)2. This cathepsin K-preferred peptide targeting sequence can enable the detection of cathepsin K enzyme activity in living, intact cells following a pre-incubation of the experimental cell population with a 10 µM concentration of CA-074. CA-074 is a specific cathepsin B inhibitor that will not inhibit intracellular cathepsin K activity. Active cathepsin K lysosomal enzyme, when present in the experimental cell population, will catalyze the hydrolysis of the two L-R target sequences from the cresyl violet fluorophore (aka Magic Red™). When the fluorophore is di-substituted with two L-R dipeptide cathepsin K targeting sequences, it is essentially non-fluorescent. Following cathepsin K-associated enzymatic hydrolysis, the two L-R peptide sequences are cleaved from the Magic Red molecule, converting it to the red fluorescent form.
As cathepsin activity progresses, more of the MR-(LR)2 is converted to the red fluorescent form of the substrate, leading to an overall increase in the red fluorescence of the cell. Because the Magic Red™ probe is cell membrane permeant, a cell lysis step is not required to perform the assay. Magic Red excites at 540-590 nm (590 nm optimal) and emits at >610nm (630 nm optimal). This signal can be detected with a fluorescence microscope or flow cytometers equipped with a green excitation laser. Each kit includes the Magic Red-(LR)2 substrate reagent, Hoechst 33342 stain, and Acridine Orange stain, allowing for cells to be simultaneously evaluated for both the presence of apoptotic nuclear morphology and cathepsin K activity in fluorescence microscopy analysis techniques. Acridine Orange dye is included to visualize lysosomal organelle structure.

* Required Fields

Cathepsin K is a lysosomal cysteine protease and a member of the papain superfamily of enzymes. Unlike cathepsins B and L, cathepsin K is expressed in a much more limited number of cell types [1]. Cathepsin K plays a vital role in its function as an efficient bone remodeling and resorption enzyme, exhibiting very potent collagenase and elastase properties [2]. It may play a similar role in the matrix remodeling process in lung tissues [3]. Although originally associated with osteoclast and ovary cells, it has subsequently been found to be naturally expressed in lung and thyroid tissues. The presence of elevated levels of cathepsin K in breast and prostate cancer cells suggests that this enzyme may facilitate the spread of these cancers [4-5]. Elevated levels of cathepsin K in the serum and synovial fluids of patients suffering from rheumatoid arthritis suggests that it plays an effector role in this disease as well [6]. This potent extracellular matrix degrading capability makes cathepsin K an important target enzyme in cancer detection, monitoring, and chemotherapy research. Assessment of elevated levels of extra-cellular cathepsins has proven to be an important prognostic indicator of cancer chemotherapy outcome.

Magic Red™ Cathepsin B detection kits from ICT provide a simple method to monitor the varying constitutive levels of generalized cathepsin B enzyme activity in whole, living cells. Browse the tabs for more information, or contact us.

  1. Drake, F.H., R. Dodds, I. James, J. Connor, C. Debouck, S. Richardson, E. Lee, D. Rieman, R. Barthlow, G. Hastings, and M. Gowen. 1996. Cathepsin K, but not cathepsin B, L, or S, is abundantly expressed in human osteoclasts. J. Biol. Chem. 271: 12511-12516.
  2. Saftig, P., E. Hunziker, V. Everts, S. Jones, A. Boyde, O. Wehmeyer, A. Suter, and K. Figura. 2000. Functions of cathepsin K in bone resorption. Adv. Exp. Med. Biol. 477: 293-303.
  3. Buhling, F., N. Waldburg, A. Gerber, C. Hackel, S. Kruger, D. Reinhold, D. Bromme, E. Weber, S. Ansorge, and T. Welte. 2000. Cathepsin K expression in human lung. Adv. Exp. Med. Biol. 477: 281-286.
  4. Podgorski, I., B.E. Linebaugh, and B.F. Sloane. 2007. Cathepsin K in bone microenvironment: link between obesity and prostate cancer. Biochem. Soc. Trans. 35: 701-703.
  5. Le Gall, C. A. Bellahcene, E. Bonnelye, J.A. Gasser, V. Castronovo, J. Green, J. Zimmermann, and P. Clezardin. 2007. A cathepsin K inhibitor reduces breast cancer induced osteolysis and skeletal tumor burden. Cancer Res. 67: 9894-9902.
  6. Skoumal, M., G. Haberhauer, G. Kolarz, G. Hawa, W. Woloszczuk, and A. Klingler. 2005. Serum cathepsin K levels of patients with longstanding rheumatoid arthritis: correlation with radiological destruction. Arthritis Res. Ther. 7: R65-R70.
Product Manuals:

Magic Red Cathepsin detection kit manual.pdf
Reagent Name: MR-(LR)2
Flourescent Label: Magic Red™ (Cresyl violet)

Sample Protocol:
Suspension Cells:

  1. Culture cells up to 1 x 10^6 cells/mL.
  2. Establish what cell environment/treatment or cell type will yield both positive and negative controls. Cathepsin K is constitutively produced in a restricted number of tissues and cell types. Care should be taken to verify whether cathepsin K is expressed in the cell type that is being investigated.
  3. Reconstitute the reagent with 50 µL DMSO to form the stock concentrate (may be frozen for future use).
  4. Dilute the stock concentrate with PBS to form the working solution.
  5. Add ~10 - 15 µL of the 31X working solution directly to a 300-500 µL aliquot of your cell culture for labeling.
  6. Incubate 30 minutes - 4 hours at 37° C in the dark.
  7. If desired: label DNA with Hoechst stain, label lysosomes with Acridine Orange, and/or fix cells.
  8. Analyze data using a fluorescence microscope or flow cytometer equipped with a green excitation laser.
Adherent Cells:
  1. Culture cell monolayers to a 80-90% confluency.
  2. Establish what cell environment/treatment or cell type will yield both positive and negative controls. Cathepsin K is constitutively produced in virtually all cell types but the level of this activity will vary by pathophysiological status.
  3. Reconstitute the reagent with 50 µL DMSO to form the stock concentrate (which can be frozen for future use).
  4. Gently remove cell culture media from flask and rinse cell culture surface once with sterile PBS. Save any loose cells for combination with trypsinized cells in Magic Red MR-(LR)2 staining steps.
  5. Add trypsin to cell monolayer and collect disassociated cells for analysis.
  6. Dilute the newly trypsinized cells in cell culture media containing 10-20% FBS and pellet down cells in this media to concentrate cells.
  7. Dilute the MR-(LR)2 stock concentrate with PBS to form the working solution.
  8. Add ~ 15 µL of the working solution directly to a 500 µL aliquot of your adherent cell culture for labeling.
  9. Incubate 30 minutes - 4 hours at 37° C in the dark.
  10. If desired: label DNA with Hoechst stain, label lysosomes with Acridine Orange, and/or fix cells.
  11. Analyze data using a fluorescence microscope or flow cytometer equipped with a green excitation laser.

  1. Hazama, R, et al. 2009. ATP-induced osteoclast function: the formation of sealing-zone like structure and the secretion of lytic granules via microtubule-deacetylation under the control of Syk. Genes Cells. 14: 871-884.

MR-LR data.

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

 

 

 

 

 

 

 

 

Target: cathepsin K
Excitation / Emission: 590 nm / 620 nm
Method of Analysis: Fluorescence Microscope
Types of Samples: cell culture, tissue
Kit Contents: Kit #939: MR-(LR)2 Reagent (one 25-test vial), Hoechst 33342 Stain (1 mL), Acridine Orange Stain (0.5mL)

Kit #940: MR-(LR)2 Reagent (one 100-test vial), Hoechst 33342 Stain (1 mL), Acridine Orange Stain (0.5mL)
Storage: 2°-8° C, Ships Overnight (Domestic), International Priority Shipping
MSDS:

MR-(LR)2

Hoechst 33342 Stain

Acridine Orange

How many tests can be run with the trial size and regular size kits?
The trial size Magic Red™ Cathepsin K detection kit (#939) provides sufficient fluorogenic substrate reagent to prepare 13mL of cell culture staining sample at 5 µM substrate concentration - or approximately 25 (0.5 mL) tests. It has been shown that these assays perform well even when used at a 5 µM staining concentration. The regular size Magic Red™ Cathepsin K detection kit (#940) provides enough fluorogenic substrate reagents to prepare 52mL of cell culture staining sample - approximately 100 tests.

What is one "test"?
One "test" is a 500 µL aliquot of cells adjusted to a concentration of 1-2 X10^6 cells/mL and analyzed on a fluorescence microscope.

How is Magic Red™ Cathepsin K substrate different from other caspase detection substrates?
The Magic Red™ substrates are the only substrate products to use the fluorogenic dye cresyl violet. Unlike the coumarin dye based substrates (AMC or AFC), cresyl violet based Magic Red substrates are cell membrane permeant and thus do not require a cell lysis step to complete.

How soon should the Magic Red-LR2-stained samples be read following addition of the substrate?
We recommend reading the cells after a 30-60 minute incubation period at 37°C, but it is possible to read the signal as early as 10 minutes post substrate addition. Each induction scenario may have different rates of Magic Red™ substrate hydrolysis and conversion to the red fluorescent product.

Call 1-800-829-3194 for technical assistance or email Technical Support: help {at} immunochemistry.com.

Related Products

Check items to add to the cart or select all