Description
Description: Magic Red Cathepsin L detection kits provide a mechanism to monitor the varying constitutive levels of generalized cathepsin L enzyme activity in whole, living cells. The assay is based on the fluorogenic substrate MR-(Phe-Arg)2 or MR-(FR)2, which contains the cresyl violet (CV) fluorophore branded as Magic Red (MR). This cathepsin L preferred peptide targeting sequence can enable the detection of cathepsin L enzyme activity in living, intact cells following a pre-incubation of the experimental cell population with a 10 uM concentration of CA-074, which is a specific cathepsin B inhibitor that will not inhibit intracellular cathepsin L activity. Active cathepsin L lysosomal enzyme, present in the experimental cell population, will catalyze the hydrolysis of the two F-R target sequences from the cresyl violet fluorophore (aka Magic Red). When the fluorophore is di-substituted with two F-R dipeptide cathepsin L targeting sequences, it is essentially non-fluorescent. Following cathepsin L-associated enzymatic hydrolysis, the two F-R peptide sequences are cleaved from the Magic Red molecule, converting it to the red fluorescent form. Because the Magic Red probe is cell membrane permeant, a cell lysis step is not required to perform the assay. As cathepsin activity progresses, more of the MR-(FR)2 is converted to the red fluorescent form of the substrate, 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). Each kit includes the Magic Red-(FR)2 substrate reagent, Hoechst 33342 stain to visualize nuclei, and Acridine Orange stain to visualize lysosomal organelle structure. This allows cells to be simultaneously evaluated for both the presence of apoptotic nuclear morphology and cathepsin L activity in fluorescence microscopy analysis techniques. Analysis by flow cytometer may also be performed if the instrument is equipped with a green laser excitation option.
Background: Cathepsin L is a lysosomal cysteine protease and a member of the papain superfamily of enzymes [1]. Cathepsin L functions as a very potent endosomal/lysosomal endopeptidase involved in the degradation and recycling of a wide range of intra- and extracellular protein substrates [2]. In addition to its lysosomal role as a processor of intracellular proteins, cathepsin L is also a key mediator of extracellular matrix breakdown leading to tumor invasion and metastasis [3]. This makes it an important target enzyme in cancer detection and chemotherapy research. Assessment of elevated levels of extracellular cathepsins has proven to be an important prognostic indicator of cancer chemotherapy outcome [4]. Magic Red Cathepsin L detection kits from ICT provide a simple method to monitor the varying constitutive levels of generalized cathepsin L enzyme activity in whole, living cells