Fluorescent Cathepsin Substrates

Cathepsins are ubiquitous lysosomal proteases that are classified according to their active site. Structural differences between various cathepsins result in variations in their substrate specificity and mechanism of inhibition. Cathepsins play an important role in the turnover of intracellular proteins and extracellular proteins via endocytosis. Extracellularly they have been implicated in tumor invasion and metastasis and, recently, as a positive mediator of apoptosis induced by gamma-interferon, Fas/APO-1, and TNF-alpha . The ability of tumor cells to invade into the extracellular matrix has been attributed to the activity of cathepsins released by tumor cells or associated with the plasma membrane of tumor cells. Benign tumors are characterized by a continuous basal lamina separating the epithelium from the stroma. However, invasive carcinomas exhibit a disrupted extracellular lamina adjacent to the invading tumor cells in the stroma. Cathepsins secreted by invading tumor cells can degrade collagen and elastin, thereby destroying the basal laminar region. In normal cells, following their synthesis, cathepsins are transported into the lysosomal compartment. However, in tumor cells, instead of being transported into the lysosomal compartment, they are secreted into the surrounding medium. The presence of cathepsins in the extracellular compartment may be employed as an ideal independent prognostic factor to determine the clinical outcome of cancer chemotherapy.

These Magic Red™ substrate-based assays are designed to detect Cathepsin protease activity within whole living cells, using fluorescence microscopy.  Magic Red™ assays are based on the cresyl violet leaving group that fluoresces upon enzyme specific peptidase activity. Staining apoptotic cells with the Magic Red™ kit can be completed within a few minutes. However, the Magic Red™ kit is used with living cells, which require periodic maintenance and cultivation several days in advance. In addition, once the proper number of cells has been cultivated, time must be allotted for the induction process. The Magic Red™ kit works with your current apoptosis protocols - induce apoptosis as you normally would and then label the cells with Magic Red™. 1. Culture cells to a density optimal for apoptosis induction according to your specific induction protocol, but not to exceed 106 cells/mL. 2. At the same time, culture a non-induced negative control cell population at the same density as the induced population for every labeling condition. 3. Induce apoptosis following your protocol (such as treating Jurkat cells with 2 mg/ml camptothecin for 3 hours). 4. Reconstitute the vial of lyophilized Magic Red™ with DMSO to form the 250X Magic Red™ stock concentrate. 5. Dilute the 25X Magic Red™ stock to the 30X working solution. 6. Stain cells by adding the 25X Magic Red™ solution. 7. Incubate for 1 hour. 8. Wash and spin cells. 9. If desired, label cells with Hoechst stain. 10. If desired, label cells with acridine orange. 11. Analyze data via fluorescence microscopy.  

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