Magic Red® Cathepsin K Assay Kit

1. Prepare samples and controls
2. Reconstitute Magic Red by adding 50 µL DMSO.
3. Dilute Magic Red 1:10 by adding 450 µL diH2O.
4. Add 20 µL Magic Red to each sample (~ 500 µL aliquot of cultured cells).
5. Incubate while protected from light.
6. Watch color start to develop within 15 minutes.
7. If desired, label with additional stains, such as Hoechst, DAPI, Acridine orange, or an antibody.
8. If desired, fix or embed cells.
9. 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.

Ratto, E;Chowdhury, SR;Siefert, NS;Schneider, M;Wittmann, M;Helm, D;Palm, W. Direct control of lysosomal catabolic activity by mTORC1 through regulation of V-ATPase assembly. Nature communications. 2022 August 17; doi: 10.1038/s41467-022-32515-6. Article

Matsumoto A, Pasut A, Matsumoto M, Yamashita R, Fung J, Monteleone E, Saghatelian A, Nakayama KI, Clohessy JG, Pandolfi PP. mTORC1 and muscle regeneration are regulated by the LINC00961-encoded SPAR polypeptide. Nature. 541(7636): 228-232. Abstract.

"Cathepsin K and L activity was monitored with MagicRed Cathepsin Assay Kit (ImmunoChemistry Technologies) according to the manufacturer’s recommendations."

Dongre A, Clements D, Fisher AJ, and Johnson SR. Cathepsin K in Lymphangioleiomyomatosis: LAM Cell-Fibroblast Interactions Enhance Protease Activity by Extracellular Acidification. Am. J. Pathol. 2017. Aug;187(8):1750-1762. doi: 10.1016/j.ajpath.2017.04.014. Epub 2017 Jun 15. Abstract.

"... peroxidase conjugate (Sigma). Cathepsin K Activity Assays. Intracellular cathepsin K activity was recorded in live cells using the Magic Red substrate (ImmunoChemistry Technologies, 2B Scientific, Bicester, UK). Cultured cells ..." Question: Can we fix cells after magic red staining and then permeabilize and incubate with LAMP1 antibody to co-stain for lysosomes?

Question: Can we fix cells after magic red staining and then permeabilize and incubate with LAMP1 antibody to co-stain for lysosomes?

Answer: We have not done any fixation procedures with this substrate but others have used a 4% paraformaldehyde treatment for 30 minutes with cold storage to extend their viewing time. Cleavage of the aspartic carboxyl frees up two primary-secondary amino groups that can react with the aldehyde functionality of the formaldehyde molecules. They don’t have the nice reactivity of a classic free amino group like you find on a lysine for instance but they are reactive and apparently fixable.

Question: My second question was if I can permeabilize cells afterfixation followed by labelling cells with LAMP-1/2 antibody to visualize lysosomes, would that work?? I am afraid of using acridine orange which cannot be used in same cells and may overlap with magic red. This LAMP labeling may help me to ascertain lysosomal vs cytoplasmic location of cathepsin since my major interest is to look for lysosomal membrane permeabilization. If you any other product related to LMP or any other advice which can be used for magic red staining in context of LMP, please do let me know.

Answer: We don’t foresee any issues with your proposed antibody labeling following Magic Red staining provided the fluorophore tag on the LAMP antibody is compatible with the fluorescence emission spectrum of the cleaved Magic Red substrate dye (cresyl violet). Acridine Orange is included in the kit to visualize lysosomal structures, however you are correct that using Magic Red substrate and Acridine Orange in the same cells should be avoided because of the overlap in emissions. We have not personally attempted to co-stain with other lysosomal markers beside Acridine Orange (AO) so unfortunately I can’t offer further advice. We intend to add an additional 2 or 3 lysosomal staining probes to our company product offering listings. But presently, we only included an example of AO staining to support the premise that our Magic Red substrates are capable of penetrating both the cell and lysosomal lipid bilayer membrane structures. Once inside lysosomal structures, our Magic Red cathepsin substrates can be converted into a fluorescent form of the substrate and detected by fluorescence microscopy techniques.

Question: I am writing you because I have a question and to let you know that I was able to fix the MagicRed CTSK probe in tissue and extend the life a couple of days. My question is, do you know the molecular weight of the MagicRed Cathepsin K probe?

Answer: The molecular weight of Magic Red Cathepsin K Substrate (MR(LR)2) is 1068.