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Magic Red Caspase-3/7 Assay Kit

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Description:

Detect caspase 3 and 7 activity in real time with the Magic Red Caspase 3/7 Assay Kit. The Magic Red reagent fluoresces upon cleavage by active caspase 3 and 7 enzymes. Analyze the fluorescent signal using fluorescence microscopy, a fluorescent plate reader, or flow cytometry.

Shelf Life:

12 months

Detection Method:

Fluorescence Microscope, Fluorescent Plate Reader, Flow Cytometer

How To Use:

1. Prepare samples and controls
2. Reconstitute Magic Red with 100 or 400 µL DMSO
3. Dilute Magic Red 1:5 with diH₂O
4. Add diluted Magic Red at 1:15-1:30 to suspension cells
5. Incubate while protected from light.
6. Watch color start to develop within 15 minutes of addition of Magic Red
7. If desired, label with additional stains, such as Hoechst, DAPI, or an antibody.
8. Analyze with a fluorescent microscope, fluorescent plate reader, or flow cytometer. Magic Red has a maximum excitation at 592 nm and emission at 628 nm. Good fluorescence images can be obtained at 510-560 nm excitation and >610 nm emission.

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

Introduction:

Caspases play important roles in apoptosis and inflammation. Detection of DEVDase enzyme activity (caspase 3 activity) is a reliable method for assessing apoptotic activity in experimental cell populations. The presence of the active form of caspase 3 & 7 in apoptotic cells causes the hydrolysis of the two DEVD target sequences from the Magic Red cresyl violet fluorophore, converting it to the fluorescent form. As apoptosis progresses and caspase activity increases, the red fluorescent signal increases. Magic Red excites at 590 nm and emits at 628 nm and can be analyzed using fluorescence microscopy, a fluorescent plate reader, or flow cytometry. Samples may also be analyzed with Acridine Orange dye or Hoechst stain to visualize lysosomal organelle structure or detect changes in nuclear morphology respectively.

Kit Contents:

25 tests
• MR-(DEVD)2 Reagent, 25-test vial.
• Hoechst 33342 Stain, 1 mL.
• Acridine Orange Stain, 1 mL.
• Kit Manual.

100 tests
• MR-(DEVD)2 Reagent, 100-test vial.
• Hoechst 33342 Stain, 1 mL.
• Acridine Orange Stain, 1 mL.
• Kit Manual.

Measurement:

590 nm / 628 nm

Sample:

Cell culture, tissue

Storage Conditions:

Store at 2-8°C.
avoid freeze/thaw cycles

Target:

Caspase 3 and 7

Testing Results:

Rat fibroblasts were seeded in a 12-well plate at 1×104 cells/mL and irradiated the following day. 23.1 µL of MR-(DEVD)2 solution was mixed with media and added. 1 hour later, 300 µL media was added and cells MR-DEVD data were photographed over 16 hours. Bright field images show increasing red intensity as caspase activity and apoptosis progressed.

It should be noted that our products are for research purposes only. Not suitable for any clinical use.

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