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Magic Red Cathepsin B Assay


Quantitate and monitor intracellular cathepsin-B activity over time in vitro. The Magic Red substrate in this assay fluoresces red upon cleavage by active cathepsin enzymes. Analyze the fluorescent signal using fluorescence microscopy or a fluorescence plate reader.

Catalog # Size Quantity Price
937 25 tests $185.00
938 100 tests $494.00
Catalog #: 937, 938 Categories: , ,

Elevated cathepsin enzyme activity in serum or the extracellular matrix can signify a number of pathological conditions. ICT’s Magic Red assay kits are used by researchers seeking to quantitate and monitor cathepsin activity in cultured cells and tissues. This assay kit detects cathepsin B activity.

To use Magic Red, add the substrate directly to the cell culture media, incubate, and analyze. Because it is cell permeant, it easily penetrates the cell membrane and the membranes of the internal cellular organelles – no lysis or permeabilization steps are required. If cathepsin enzymes are active, the Magic Red substrate is cleaved and the cresyl violet fluorophore will become fluorescent upon excitation. As enzyme activity progresses and more Magic Red substrate is cleaved, the signal will intensify, allowing researchers to watch the color develop over time. Samples can be analyzed by fluorescence microscopy or with a fluorescence plate reader. Hoechst 33342 and Acridine Orange are included in the kit to detect nuclear morphology and lysosomal organelle structure, respectively.

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 (480 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 33342, DAPI, or an antibody.
8. If desired, fix 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.

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

• Reagent name: MR-RR2
• Target: Cathepsin-B
• Excitation/Emission: 592 nm / 628 nm
• Method of Analysis: Fluorescence Plate Reader, Fluorescence Microscope
• Types of Samples: Cell culture
• Storage: 2-8°C
• Shipping: Ships overnight (domestic), International Priority Shipping

Kit 937: 25 Tests
• Magic Red Substrate (MR-RR2), 1 25 Test Vial, #6133
• Hoechst 33342, 1 mL, #639
• Acridine Orange, 0.5 mL, #6130
• Kit Manual

Kit 938: 100 Tests
• Magic Red Substrate (MR-RR2), 1 100 Test Vial, #6134
• Hoechst 33342, 1 mL, #639
• Acridine Orange, 0.5 mL, #6130
• Kit Manual

Hover S, Foster B, Fontana J, Kohl A, Goldstein SAN, Barr JN, Mankouri J. Bunyavirus requirement for endosomal K+ reveals new roles of cellular ion channels during infection. PLoS Pathog. 2018. Jan 19;14(1):e1006845. doi: 10.1371/journal.ppat.1006845. eCollection 2018 Jan. Full text

“Cells were treated with 10 μM AG4 and 2 μg/ml Texas Red labelled EGF, Magic Red cathepsin B dye (ImmunoChemistry Technologies; ex.max 590 nm, em.max 620 nm) or 10 μg/ml pHRodo Red Dextran (Life Technologies; ex.max 560 nm, em.max 585 nm) for the indicated timepoints at 37°C.”

Festa BP, Chen Z, Berquez M, Debaix H, Tokonami N, Prange JA, Hoek GV, Alessio C, Raimondi A, Nevo N, Giles RH, Devuyst O, Luciani A. Impaired autophagy bridges lysosomal storage disease and epithelial dysfunction in the kidney. Nat Commun. 2018. Jan 11;9(1):161. doi: 10.1038/s41467-017-02536-7. Full text

“The detection of lysosomal activity was performed in live mPTCs using Bodipy-FL-PepA (P12271, Thermo Fischer Scientific) or Magic Red-(RR)2 substrate (MR-CtsB; 938, Immunochemistry Technologies) according to the manufacturer’s specifications. The cells were pulsed with 1 μM Bodipy-FL-Pepstatin A or with 1 μM Magic Red-(RR)2 in Live Cell Imaging (A14291DJ,…”

Kratschmer C, Levy M. Targeted Delivery of Auristatin Modified Toxins to Pancreatic Cancer Using Aptamers. Molecular Therapy-Nucleic Acids. 2017. 10:227-236 March 2018. doi.org/10.1016/j.omtn.2017.11.013. Full text

“Aptamers were added to cells at a final concentration of 500 μM, and Magic Red cathepsin B assay (ImmunoChemistry Technologies, Bloomington, MN) was added to cells at the manufacturer’s recommended concentration.”

Fujita N, Huang W, Lin TH, Groulx JF, Jean S, Nguyen J, Kuchitsu Y, Koyama-Honda I, Mizushima N, Fukuda M, Kiger AA. Genetic screen in Drosophila muscle identifies autophagy-mediated T-tubule remodeling and a Rab2 role in autophagy. Elife. 2017. Full text.

“The following reagents were used: Alexa Fluor 546 Phalloidin (1.0 U/ml; Invitrogen/Thermo Fisher Scientific, Waltham, MA), LysoTracker Red DND-99 (1:5,000; Thermo Fisher Scientific, Waltham, MA) and Magic Red Cathepsin B Assay (ImmunoChemistry Technologies, Bloomington, MN).”

Hamanaka T, Nishizawa K, Sakasegawa Y, Oguma A, Teruya K, Kurahashi H, Hara H, Sakaguchi S, Doh-Ura K. Melanin or melanin-like substance interacts with the N-terminal portion of prion protein and inhibits abnormal prion protein formation in prion-infected cells. J Virol. 2017. Abstract.

“Activity was visualized in cells by Magic Red cathepsin B substrate 161 MR-RR2 (Immunochemistry Technologies, Bloomington, MN) and then analyzed by fluorescence microscopy or fluorescence plate reader, according to the manufacturer’s … ”

Kang HT, Park JT, Choi K, Kim Y, Choi HJC, Jung CW, Lee YS, Chul Park S. Chemical screening identifies ATM as a target for alleviating senescence. Nat Chem Biol. 2017. Jun;13(6):616-623. Epub 2017 Mar 27. Abstract.

“Cathepsin activity was determined with MagicRed cathepsin B (CTSB) and cathepsin L (CTSL) assay kits (MR-CTSB and MR-CTSL, respectively; ImmunoChemistry Technologies), according to the manufacturer’s instructions.”

Sakamaki JI, Wilkinson S, Hahn M, Tasdemir N, O’Prey J, Clark W, Hedley A, Nixon C, Long JS, New M, Van Acker T, Tooze SA, Lowe SW, Dikic I, and Ryan KM. Bromodomain Protein BRD4 Is a Transcriptional Repressor of Autophagy and Lysosomal Function. Mol. Cell. 2017. May 18;66(4):517-532.e9. doi: 10.1016/j.molcel.2017.04.027. Full text.

“To measure lysosomal Cathepsin B activity, cells were incubated with Magic Red CathepsinB (ImmunoChemistry Technologies, Cat#: 938) for 1 hr according to the manufacturer’s instructions.”

Ríos-Luci C, García-Alonso S, Díaz-Rodríguez E, Nadal-Serrano M, Arribas J, Ocaña A, Pandiella A. Resistance to the antibody-drug conjugate T-DM1 is based in a reduction in lysosomal proteolytic activity. Cancer Res. 2017 Jul 7. doi: 10.1158/0008-5472.CAN-16-3127. [Epub ahead of print]. Abstract.

“Cells were collected and acquired using an Accuri C6 Flow Cytometer to quantify FL2H-positive cells. Cathepsin B enzymatic activity in live cells was measured using Magic Red Cathepsin B detection kit (Immunochemistry Technologies, LLC, Bloomington, USA), following the manufacturer’s instructions.”

Qian Q, Zhang Z, Orwig, A, Chen S, Ding WX, Xu Y, Kunz RC, Lind NRL, Stamler JS, Yang L. S-nitrosoglutathione Reductase Dysfunction Contributes to Obesity-Associated Hepatic Insulin Resistance via Regulating Autophagy. Diabetes. 2017. Oct 26. pii: db170223. doi: 10.2337/db17-0223. [Epub ahead of print]. Abstract

” …The HexB activity assay was performed as described by Vaidyanathan et al. (28). The CTSB assay was performed using a CTSB Detection Kit (ImmunoChemistry Technologies), and lysosomal pH was measured using LysoSensr Green DND-189 (Thermo Fisher Scientific …”

Chen J, Ou Y, Li Y, Hu S, Shao LW, Liu Y. Metformin extends C. elegans lifespan through lysosomal pathway. Elife. 2017.Oct 13;6. pii: e31268. doi: 10.7554/eLife.31268. Full Text

“Magic Red stain (ImmunoChemistry Technologies #938, Bloomington, Minnesota) can be cleaved by cathepsin B, and generate red fluorescent substrate in functional lysosomes.”