綠色組織蛋白酶 B 測定

Green Cathepsin B Assay 允許研究人員在體外使用活的全細胞檢測和監測細胞內組織蛋白酶的活性。體外測定採用Rhodamine 110-(RR)2，其在被活性組織蛋白酶切割後發出綠色螢光。使用流式細胞儀或螢光顯微鏡分析樣品。

Background 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.

Reagent Name R110-(RR)2

Target Cathepsin B

Excitation / Emission 500 nm / 525 nm

Method of Analysis Flow cytometry, Fluorescence microscope

Sample Type Cell culture

Storage R110-(RR)2 at ≤-20°C, other components at 2-8°C

Country of Origin United States

Protocols

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 Contents

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

Citations (1)

Mondal, A;NeMoyer, R;Vora, M;Napoli, L;Syed, Z;Langenfeld, E;Jia, D;Peng, Y;Gilleran, J;Roberge, J;Rongo, C;Jabbour, SK;Langenfeld, J. Bone morphogenetic protein receptor 2 inhibition destabilizes microtubules promoting the activation of lysosomes and cell death of lung cancer cells. Cell communication and signaling : CCS. 2021 September 25; doi: 10.1186/s12964-021-00743-w. Full Text