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Magic Red® Fluorescent Caspase-3/7 Assay Kit
Magic Red® Fluorescent Caspase-3/7 Assay Kit
- 中文名称：
- Magic Red® Fluorescent Caspase-3/7 Assay Kit
- 英文名称：
- Magic Red® Fluorescent Caspase-3/7 Assay Kit
- 品牌：
- Immuno Chemistry
- 货号：
- 935
- 规格：
- 25 Tests
- 保存建议：
- 2-8°C
- 背景资料：
- Our Magic Red® Fluorescent Caspase-3/7 Assay Kit enables researchers to detect and monitor in vitro apoptosis over time via intracellular caspase activity. The Magic Red® (MR) reagent is a non-cytotoxic substrate that fluoresces upon cleavage by active caspase-3/7 enzymes. It measures the intracellular process of apoptosis instead of a side effect, such as the turn-over of phosphatidyl serine.To use Magic Red®, add the substrate directly to the cell culture media, incubate, and analyze. Because MR 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 caspase-3/7 enzymes (DEVDases) are active, they will cleave the intact (quenched) substrate and release the cresyl violet fluorophore, which will greatly enhance the cresyl violet fluorescence potential. The red fluorescent product will often aggregate inside lysosomes (Figures 2 and 4); caspases are not lysosomal enzymes. As protease activity progresses and more MR substrate is cleaved, the red fluorescent signal potential will intensify, enabling researchers to watch it increase over time (Figure 3) and quantify apoptosis (Figures 7 and 10-12).There is no interference from pro-caspases or inactive forms of the enzymes. If the treatment or experimental condition is causing cell death via apoptosis, apoptotic cells will have elevated levels of caspase-3/7 activity relative to non-apoptotic or negative control cells. Up-regulation and initiation of the caspase enzyme cascade is the central driving force behind apoptosis. Although a number of other intracellular enzyme families, including the cathepsins, calpains, and granzymes, participate in the cell breakdown mechanism, the caspase cascade occupies the central effector role in the cell suicide process. Like other intracellular proteases, caspases are initially synthesized as inactive zymogen precursors that can be rapidly activated upon auto and heterologous enzymatic processing at specific sites containing an aspartic acid. Caspase-3 is the predominant effector caspase in apoptosis with few exceptions, such as MCF-7 cells, which are deficient in caspase-3 (Figures 2, 4, and 5).Caspase enzymes cleave proteins and are classified as cysteine pro- teases based on the mechanism of substrate hydrolysis at their active site. Caspases specifically recognize a 3 or 4 amino acid sequence which must include an aspartic acid residue (D) in the P1 position. This C–terminal residue is the target for the cleavage reaction at the carbonyl end. Our MR caspase-3/7 substrate contains a 4 amino acid sequence, aspartylglutamylvalanylaspartic acid (DEVD), which is the optimal target sequence for caspases 3 and 7.Two copies of this sequence are coupled to a photostable red fluorophore, cresyl violet, to create the Magic Red caspase-3/7 substrate MR-(DEVD)2. In the intact MR(DEVD)2 substrate, the fluorescence has been quenched. Maximum fluorescence potential is achieved upon cleavage of both DEVD side chains by activated caspases 3 and 7.MR has been shown to work in human, rat, and mouse cells, among other species. A baseline level of DEVDase activity is present in all cell lines. Apoptotic cells will fluoresce red and have pronounced red lysosomes and mitochondria. Healthy and non-apoptotic cells will exhibit very low levels of background red fluorescence evenly distributed throughout the cell (Figures 2-4, 7-9). This background level of substrate activity could be the result of constitutively synthesized serine proteases that target analogous aspartic acid sequences for hydrolysis. Cells in more advanced stages of apoptosis, containing peak levels of DEVDase activity, will display brighter red fluorescence than cells in earlier stages of apoptosis.The MR fluorophore, cresyl violet, fluoresces red when excited at 550-590 nm. The red fluorescent signal can be monitored with a fluorescence microscope (Figures 2-4, 7-9), plate reader (Figure 10), or flow cytometer (Figures 11 and 12). It has an optimal excitation of 592 nm and emission of 628 nm in aqueous solutions. At these higher excitation wavelengths, the amount of cell-mediated auto-fluorescence is minimal. The excitation peak is rather broad, allowing good excitation efficiency at 540-560 nm. In flow cytometry applications, optimal results can be achieved using an orange 594 nm laser and a 685/35 filter pairing, or similar. However, good results have also been generated using a more common 640 nm red laser excitation with a 675/25 filter pairing.
- 货期：
- 4-6周
- 样本类型：
- Cell culture, tissue
- 检测方法：
- Fluorescence Microscope, Fluorescent Plate Reader, Flow Cytometer
- 免责声明：
- *本产品仅供科研实验使用，不得用于临床诊断。*
- 说明书：
The Magic Red® reagent fluoresces upon cleavage by active caspase 3 and caspase 7 enzymes, enabling real-time detection of activity. Analyze the fluorescent signal using fluorescence microscopy, a fluorescent plate reader, or flow cytometry.
