神奇紅組織蛋白酶 B 檢測試劑盒

在體外隨時間推移定量和監測細胞內組織蛋白酶 B 的活性。該測定中的神奇紅底物在被活性組織蛋白酶切割後發出紅色螢光。使用螢光顯微鏡或螢光盤式分析儀分析螢光訊號。

Background

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.

Reagent Name

MR-RR2

Target

Cathepsin B

Excitation / Emission

592 nm / 628 nm

Method of Analysis

Fluorescence Plate Reader, Fluorescence Microscope

Sample Type

Cell culture

Storage

2-8°C

Country of Origin

United States

Protocols

1. Prepare samples and controls

2. Reconstitute Magic Red by adding DMSO.

3. Dilute Magic Red 1:10 by adding diH2O.

4. Add 20 µL Magic Red to each sample (~ 500 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, 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.

****If working with adherent cells, please see the manual for additional protocols.

Kit Contents

Kit#937:

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

Citations (22)

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Frequently Asked Questions

Question: When you dilute the stock concentration 1/10 in H2O and aliquot is for future use, how long can this be stored? I see that the stock concentration in DMSO can be stored for 6 months. Answer: Once the stock solution is diluted 1:10 in diH2O we recommend using within 15 minutes to prevent hydrolysis of the Magic Red substrate. Non-specific hydrolysis would lead to fluorescence not associated with enzymatic cleavage of the substrate. We do not recommend attempting to aliquot or store the diH2O-diluted substrate solution for future use. Yes, that’s correct. The DMSO-reconstituted Magic Red stock can be stored frozen for up to 6 months protected from light and thawed more than twice during that time.