Green Fluorescent Cathepsin B Assay

The quenched substrate Rhodamine 110-(RR)2, fluoresces green upon cleavage by active cathepsins, allowing the monitoring of intracellular cathepsin activity in vitro using live whole cells. Analyze using flow cytometry or fluorescence microscopy.

Figure 1. R110-(RR)2 was reconstituted in DMSO, diluted in an aqueous buffer, and then spiked with cathepsin B enzyme and incubated at 37°C for 1 hour. After incubation, fluorescence was analyzed on a Molecular Devices M5e plate reader. The excitation spectrum (black) was generated using an emission of 590 nm. The emission spectrum (green) was generated using an excitation of 460 nm.
Figure 2. Jurkat cells were treated with the cathepsin inhibitors CA-074 Me (blue) and E-64d (gray), or were untreated (green) and then stained with R110-(RR)2. Samples were then analyzed analyzed by flow cytometry (488 nm excitation and a 530/30 emission filter). Treatment with CA-074 Me or E-64d decreased the fluorescence signal detected compared to the untreated control, which displayed a baseline level of cathepsin activity. Data courtesy of Dr. Kristi Strandberg (ICT 231:40-42).
Figure 3. THP-1 monocytes were either untreated to show baseline cathepsin activity (green) or were treated with the cathepsin inhibitor CA-074 Me (blue) to reduce cathepsin activity. Cells were stained with R110-(RR)2 and analyzed by flow cytometry (488 nm excitation and 530/30 emission filter). Treatment with CA-074 Me decreased the fluorescence signal detected compared to the untreated cell population. Data courtesy of Dr. Kristi Strandberg (ICT 231:47-50).
Figure 4. Jurkat cells were treated with the cathepsin inhibitor, E-64d (lower), or were untreated (upper). Cells were then stained with R110-(RR)2, followed by Hoechst 33342, and then mounted onto coverslips and imaged. Intracellular localization of the hydrolyzed (fluorescent) R110 product was detected using EGFP (Ex 470/30, Em 530/50) and DAPI (Ex 375/28, Em 460/50) LED filter cubes at 20X. The overlay includes a brightfield image. Data courtesy of Dr. Kristi Strandberg (ICT 231:65-68).
Figure 5. THP-1 cells were treated with PMA (5 ng/mL for 48 hours) to become macrophage-like, and then exposed to the cathepsin inhibitor, CA-074 Me (lower), or were untreated (upper). Cells were then stained with R110-(RR)2 and Hoechst 33342. Cells were imaged using EGFP (Ex 470/30, Em 530/50) and DAPI (Ex 375/28, Em 460/50) LED filter cubes at 20X. Data courtesy of Dr. Kristi Strandberg (ICT 231:69).
Figure 1. R110-(RR)2 was reconstituted in DMSO, diluted in an aqueous buffer, and then spiked with cathepsin B enzyme and incubated at 37°C for 1 hour. After incubation, fluorescence was analyzed on a Molecular Devices M5e plate reader. The excitation spectrum (black) was generated using an emission of 590 nm. The emission spectrum (green) was generated using an excitation of 460 nm.
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Figure 1. R110-(RR)2 was reconstituted in DMSO, diluted in an aqueous buffer, and then spiked with cathepsin B enzyme and incubated at 37°C for 1 hour. After incubation, fluorescence was analyzed on a Molecular Devices M5e plate reader. The excitation spectrum (black) was generated using an emission of 590 nm. The emission spectrum (green) was generated using an excitation of 460 nm.
Figure 2. Jurkat cells were treated with the cathepsin inhibitors CA-074 Me (blue) and E-64d (gray), or were untreated (green) and then stained with R110-(RR)2. Samples were then analyzed analyzed by flow cytometry (488 nm excitation and a 530/30 emission filter). Treatment with CA-074 Me or E-64d decreased the fluorescence signal detected compared to the untreated control, which displayed a baseline level of cathepsin activity. Data courtesy of Dr. Kristi Strandberg (ICT 231:40-42).
Figure 3. THP-1 monocytes were either untreated to show baseline cathepsin activity (green) or were treated with the cathepsin inhibitor CA-074 Me (blue) to reduce cathepsin activity. Cells were stained with R110-(RR)2 and analyzed by flow cytometry (488 nm excitation and 530/30 emission filter). Treatment with CA-074 Me decreased the fluorescence signal detected compared to the untreated cell population. Data courtesy of Dr. Kristi Strandberg (ICT 231:47-50).
Figure 4. Jurkat cells were treated with the cathepsin inhibitor, E-64d (lower), or were untreated (upper). Cells were then stained with R110-(RR)2, followed by Hoechst 33342, and then mounted onto coverslips and imaged. Intracellular localization of the hydrolyzed (fluorescent) R110 product was detected using EGFP (Ex 470/30, Em 530/50) and DAPI (Ex 375/28, Em 460/50) LED filter cubes at 20X. The overlay includes a brightfield image. Data courtesy of Dr. Kristi Strandberg (ICT 231:65-68).
Figure 5. THP-1 cells were treated with PMA (5 ng/mL for 48 hours) to become macrophage-like, and then exposed to the cathepsin inhibitor, CA-074 Me (lower), or were untreated (upper). Cells were then stained with R110-(RR)2 and Hoechst 33342. Cells were imaged using EGFP (Ex 470/30, Em 530/50) and DAPI (Ex 375/28, Em 460/50) LED filter cubes at 20X. Data courtesy of Dr. Kristi Strandberg (ICT 231:69).

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Product Manual
Safety Data Sheet

SKU: 9152

Size: 100 Tests
Price:
$560.00
Ships: 1-2 business days
  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.
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

    • Bulk Order Green Fluorescent Cathepsin B Assay

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