Green Cathepsin B Assay


ICT’s Green Cathepsin B Assay allows researchers to detect and monitor intracellular cathepsin activity over time in vitro using live whole cells.  This in vitro assay employs the quenched substrate Rhodamine 110-(RR)2, which fluoresces green upon cleavage by active cathepsin enzymes. Analyze samples using flow cytometry or fluorescence microscopy.

Catalog # Product Size Quantity Price
9151 25 Tests $185.00
9152 100 Tests $494.00
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Cathepsins are a group of protease enzymes typically found in the lysosome. Cathepsin B plays important roles in many cellular functions (like intracellular protein degradation, antigen processing, etc.). Elevated cathepsin activity is associated with many disease states such as cancer, Alzheimer’s disease, and so on.

The Green Cathepsin B Assay contains Rhodamine 110 Cathepsin B Substrate, which is a non-cytotoxic and cell membrane permeant substrate that fluoresces green upon cleavage by active cathepsins. To use Rhodamine 110-(RR)2, add the substrate directly to the cell culture medium, incubate, and analyze. Because the substrate 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 active cathepsin enzymes are present, the quenched Rhodamine 110-(RR)2 substrate is cleaved, resulting in an increase in green fluorescence signal. Samples can be analyzed by flow cytometry or fluorescence microscopy. Hoechst 33342 is included in the kit to label nuclei.

1. Dilute 10X Cellular Assay Buffer 1:10 with diH2O.
2. Prepare samples and controls in fresh cell culture medium or 1X Cellular Assay Buffer.
3. Reconstitute R110-(RR)2 with 50 µL DMSO.
4. Dilute R110-(RR)2 1:5 by adding 200 µL PBS.
5. Add diluted R110-(RR)2 to each sample at 1:50 (e.g., add 10 µL of diluted R110-(RR)2 to 490 µL of sample).
6. Incubate while protected from light.
7. If desired, label with additional stains, such as Hoechst 33342, DAPI, or an antibody.
8. Analyze with a flow cytometer or fluorescence microscope. R110-(RR)2 excites at 500 nm and emits at 525 nm.

Cathepsin activity in THP-1 monocytes. Intracellular cathepsin activity was detected in THP-1 monocytes that were either untreated to show baseline cathepsin activity (green histogram) or were treated with the cathepsin inhibitor CA-074 Me (blue histogram) for 3 hours to reduce cathepsin activity. After treatment, cell populations were stained for 1 hour at 37°C with R110-(RR)2 . After incubation with the R110-(RR)2 substrate, the samples were analyzed by flow cytometry with an Attune NxT flow cytometer equipped with a blue laser (488 nm excitation and 530/30 emission filter). An overlay of the histograms is shown below. Treatment with CA-074 Me decreased the fluorescence signal detected compared to the untreated cell population.
R110-RR2 monocyte figure f

Cathepsin activity in THP-1 macrophages. Intracellular cathepsin activity was detected in THP-1 cells. THP-1 cells were seeded onto chamber slides and were then treated with PMA (5 ng/mL for 48 hours) to become macrophage-like. After 48 hours, media containing PMA was replaced with fresh media, and cells were allowed to recover for 4 days, and then were exposed to the cathepsin inhibitor, CA-074 Me for 3 hours (lower row of images), or were untreated (upper row of images). Following treatment, samples were stained with R110-(RR)2 for 1 hour at 37°C and Hoechst 33342 for 15 minutes at room temperature, and then were imaged. Intracellular localization of the hydrolyzed (fluorescent) R110 product was detected using a Logos iRiS Digital Cell Imaging System equipped with a EGFP (Ex 470/30, Em 530/50) and a DAPI (Ex 375/28, Em 460/50) LED filter cubes at 20X. The images below show untreated cells (upper row of images) and inhibitor-treated cells (lower row of images) stained with R110-(RR)2 (left most images), Hoechst 33342 (middle images), or an overlay of both the EGFP and DAPI channels (rightmost images).
R110-RR2 macrophage figure

• Reagent name: R110-(RR)2
• Target: Cathepsin B
• Excitation/Emission: 500 nm / 525 nm
• Method of Analysis: Flow cytometer, Fluorescence Microscope
• Types of Samples: Cell culture
• Storage: Store R110-(RR)2 at ≤-20°C. Store all other reagents at 2-8°C
• Shipping: Ships overnight (domestic), International Priority Shipping

Kit 9151: 25 Tests
• 1 vial of Rhodamine 110-(RR)2 substrate [R110-(RR)2], 25 Tests, #6691
• 1 vial of Hoechst 33342 Stain, 200 µg/mL (1 mL), #639
• 1 bottle of 10X Cellular Assay Buffer (15 mL), #6694
• Kit Manual

Kit 9152: 100 Tests
• 4 vials of Rhodamine 110-(RR)2 substrate [R110-(RR)2], 25 Tests, #6691
• 1 vial of Hoechst 33342 Stain, 200 µg/mL (1 mL), #639
• 1 bottle of 10X Cellular Assay Buffer (60 mL), #6695
• Kit Manual


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