Magic Red Cathepsin K Assay Kit

Magic Red Cathepsin K Assay Kit

Cat. Num. AAA258032

• Synonyms: Magic Red Cathepsin K; Magic Red Cathepsin K Assay Kit; Magic Red Cathepsin K assay kit

• Reagents: MR-(LR)2
• Suspension Cells: Culture cells up to 1 x 10^6 cells/mL. 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. Reconstitute the Magic Red reagent with DMSO to form the stock concentrate (which can be frozen for future use). Dilute the Magic Red stock concentrate 1:10 with diH2O to form the working solution. Add ~10 - 15 uL of the working solution directly to a 300-500 uL aliquot of your cell culture for labeling. The working solution is used at approximately 1:15-1:26 to label suspension cells. Incubate 30 minutes - 4 hours at 37 degree C in the dark. If desired label DNA with Hoechst stain, and/or fix cells. Unstained cells may be labeled with Acridine Orange to reveal the lysosomes. Analyze data using a fluorescence microscope, fluorescence plate reader, or flow cytometer.
• Adherent Cells: Culture cell monolayers to 80-90% confluency. 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. 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. Add trypsin to cell monolayer and collect disassociated cells for analysis. Dilute the newly trypsinized cells in cell culture media containing 10-20% FBS and pellet down cells in this media to concentrate cells. Reconstitute the Magic Red reagent with DMSO to form the stock concentrate (which can be frozen for future use). Dilute the Magic Red stock concentrate 1:10 with diH2O to form the working solution. Add ~ 15 uL of the working solution directly to a 500 uL aliquot of your adherent cell culture for labeling. The working solution is used at approximately 1:26 to label adherent cells. Incubate 30 minutes - 4 hours at 37 degree C in the dark. If desired label DNA with Hoechst stain, and/or fix cells. Unstained cells may be labeled with Acridine Orange to reveal the lysosomes. Analyze data using a fluorescence microscope or fluorescence plate reader.
• Target: Cathepsin K
• Excitation / Emission: 590 nm / 620 nm
• Method of Analysis: Fluorescence Microscope
• Types of Samples: Cell Culture, Tissue
• Preparation and Storage: Store at 2-8 degree C

Testing Data

(Cathepsin K-positive THP-1 cellsfluoresce red after staining withMR-(LR)2. Red fluorophoreconcentrates inside lysosomes (Dr.Brian W. Lee, ICT).)

Related Product Information for Magic Red Cathepsin K assay kit

Description: Magic Red Cathepsin K Assay Kits provide a simple method to monitor the varying constitutive levels of generalized cathepsin K enzyme activity in whole, living cells. This Cathepsin K detection assay is based on the cell permeant, fluorogenic substrate MR-(Leu-Arg)2, aka MR-(LR)2, which becomes fluorescent after cathepsin K-associated enzymatic hydrolysis of the LR target sequences from the fluorophore. As cathepsin activity progresses, more of the MR-(LR)2 substrate is converted to its red fluorescent form, leading to an overall increase in the red fluorescence of the cell. 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, lysosomal organelle structure, and cathepsin K activity in fluorescence microscopy analysis techniques.

Background: Assess Cathepsin K Activity with Magic Red Cathepsin K Assay Kits - 25 tests - 100 tests 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. Assay Principle: Magic Red Cathepsin K Assay is based on the cresyl violet (CV) fluorogenic substrate CV-(Leu-Arg)2 (aka CV-(LR)2, MR-(Leu-Arg)2, MR-(LR)2). Cresyl violet is a red fluorophore, branded Magic Red; when di-substituted with two peptides, it is essentially non-fluorescent. Leucine-Arginine is a peptide sequence preferentially targeted by activated cathepsin K enzymes. With this cathepsin K-preferred peptide sequence (LR), the Magic Red fluorogenic substrate enables the detection of cathepsin K enzyme activity in living, intact cells. A pre-incubation is recommended with a 10 uM concentration of CA-074, a specific cathepsin B inhibitor that will not inhibit intracellular cathepsin K activity. Active cathepsin K lysosomal enzyme, when present in the cell population, will catalyze the hydrolysis of the two L-R target sequences linked to the Magic Red fluorophore. Following cathepsin K-associated enzymatic hydrolysis, the two L-R peptide sequences are cleaved from the Magic Red fluorogenic 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, lysosomal organelle structure, and cathepsin K activity in fluorescence microscopy analysis techniques. Magic Red Cathepsin K detection kits from MyBioSource provide a simple method to monitor the varying constitutive levels of generalized cathepsin K enzyme activity in whole, living cells.

Product Categories/Family for Magic Red Cathepsin K assay kit

Apoptosis Assays/Cathepsin Assays

References

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. 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. 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. 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. 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. 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.