Magic Red™ Caspase 3 & 7 Assay Kit

Detect Caspase 3 and 7 Activity in Real Time with Magic Red

Magic Red™ -(DEVD)2 caspase 3/7 fluorogenic substrate is primarily targeted by caspases -3 and -7 of the apoptotic protease cascade. Caspase-3 is the most important caspase of the executioner caspase group that also includes caspases -6 and -7 [1]. The inactive zymogen form of caspase-3 is efficiently processed by any of the initiator caspases (caspase-8, caspase-9, and caspase-10). All executioner caspases will target the various cytoskeletal structural proteins as well as PARP and ICAD leading to the morphological and biochemical changes that drive the apoptotic process [2]. Detection of DEVDase enzyme activity is a reliable method for assessing apoptotic activity in experimental cell populations [3].

Magic Red™ Caspases 3 & 7 detection kits measure apoptosis-associated DEVDase enzyme activity in living, intact cells. The presence of the active form of caspases 3&7 in apoptotic cells causes the hydrolysis of the two DEVD target sequences from the cresyl violet fluorophore (Magic Red™), converting it to the fluorescent form. When the cresyl violet dye is di-substituted with two DEVD caspase-3/7 target sequences, the reagent is essentially non-fluorescent.  As apoptosis progresses and caspase activity increases, the red fluorescent signal increases. Magic Red™ (cresyl violet) excites at 540-590 nm (590 nm optimal) and emits at >610nm (630 nm optimal). Because the Magic Red™ substrate probe is cell membrane permeant, a cell lysis step is not required to perform the assay.

1. Elmore, S. 2007. Apoptosis: A review of programmed cell death. Toxicol. Pathol. 35: 495-516.
2. McStay, G.P., G.S. Salvesen, and D.R. Green. 2008. Overlapping cleavage motif selectivity of caspases: implications for analysis of apoptotic pathways. Cell Death Differ. 15: 322-331.
3. Lee, B.W., G.L. Johnson, S.A. Hed, Z. Darzynkiewicz, J.W. Talhouk, and S. Mehrotra. 2003. DEVDase detection in intact apoptotic cells using the cell permeant fluorogenic substrate, (z-DEVD)2-cresyl violet. BioTechniques 35: 1080-1085.

Sample Protocol:
Suspension Cells:

1. Culture cells up to 1 x 10^6 cells/mL.
2. Induce apoptosis following your protocol, and create positive and negative controls.
3. Reconstitute the reagent with DMSO to form the stock concentrate (which can be frozen for future use).
4. Dilute the stock concentrate with diH2O to form the working solution.
5. Add ~10 - 15 µL of the 31X working solution directly to a 300-500 µL aliquot of your cell culture for labeling.
6. Incubate 30 minutes - 4 hours at 37° C in the dark.
7. If desired: label DNA with Hoechst stain, label lysosomes with Acridine Orange, and/or fix cells.
8. Analyze data using a fluorescence microscope, fluorescence plate reader, or flow cytometer equipped with a green excitation laser.

1. Culture cell monolayers to a 80-90% confluency.
2. Induce apoptosis following your protocol, and create positive and negative controls.
3. Reconstitute the reagent with DMSO to form the stock concentrate (which can be frozen for future use).
4. Gently remove cell culture media from flask and rinse cell culture surface once with sterile PBS. Save any loose cells for combination with trypsinized cells in Magic Red MR-(DEVD)2 staining steps.
5. Add trypsin to cell monolayer and collect disassociated cells for analysis.
6. Dilute the newly trypsinized cells in cell culture media containing 10-20% FBS and pellet down cells in this media to concentrate cells.
7. Dilute the MR-(DEVD)2 stock concentrate with diH2O to form the working solution.
8. Add ~ 15 µL of the working solution directly to a 500 µL aliquot of your adherent cell culture for labeling.
9. Incubate 30 minutes - 4 hours at 37° C in the dark.
10. If desired: label DNA with Hoechst stain, label lysosomes with Acridine Orange, and/or fix cells.
11. Analyze data using a fluorescence microscope, fluorescence plate reader, or flow cytometer equipped with a green excitation laser.

1. Lee, BW, et al. 2003. DEVDase detection in intact apoptotic cells using the cell permeant fluorogenic substrate, (z-DEVD)2-cresyl violet. BioTechniques, 35(5): 1080-1085.
2. Khan, N, et al. 2008. Fisetin, a novel dietary flavonoid, causes apoptosis and cell cycle arrest in human prostate cancer LNCaP cells. Carcinogenesis, 29: 1049-1056.
3. Perros, F, et al. 2008. Platelet-derived Growth Factor Expression and Function in Idiopathic Pulmonary Arterial Hypertension. Am. J. Resp. Crit. Care, 178: 81-88.
4. Morelli, MB, et al. 2008. Characterization, Expression, and Functional Activity of Pituitary Adenylate Cyclase-Activating Polypeptide and Its Receptors in Human Granulosa-Luteal Cells. J. Clin. Endocr. Metab., 93: 4924 - 4932.
5. Jang, SW, et al. 2010. A selective TrkB agonist with potent neurotrophic activities by 7,8-dihydroxyflavone. PNAS, 107: 2687-2692.

View a time-lapse movie of caspase-3/7 activation in cultured rat fibroblasts. Data courtesy of Dr. Martin Purschke, MA General Hospital. Windows Media Player or similar required to play the .avi file.

Rat fibroblasts were seeded in a 12-well plate at 1x104 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 were
photographed
over 16 hours.
Bright field images
show increasing
red intensity
as caspase
activity and
apoptosis
progressed (Dr. Martin Purschke, Massachusetts General Hospital).

How many tests can be run with the trial size and regular size kits?
The trial size Magic Red™ Caspases 3 & 7 kit (#935) provides sufficient fluorogenic substrate reagent to prepare 7.5mL of cell culture staining sample at 20 µM substrate concentration - or approximately 25 (0.3 mL) tests. It has been shown that these assays perform well even when used at a 5 µM staining concentration. The regular size Magic Red™ Caspases 3 & 7 kit (#936) provides enough fluorogenic substrate reagents to prepare 30mL of cell culture staining sample - approximately 100 tests.

What is one "test"?
One "test" is a 300 µL aliquot of cells adjusted to a concentration of 1-2 X10^6 cells/mL and analyzed on a fluorescence plate reader or microscope.

How is Magic Red™ caspase 3/7 substrate different from other caspase detection substrates?
The Magic Red™ substrates are the only substrate products to use the fluorogenic dye cresyl violet. Unlike the coumarin dye based substrates (AMC or AFC), cresyl violet based Magic Red substrates are cell membrane permeant and thus do not require a cell lysis step to complete.

How soon should the Magic Red-DEVD-stained samples be read following addition of the substrate?
We recommend reading the cells after a 30-60 minute incubation period at 37°C if using a fluorescence plate reader and as early as 10-15 minutes post substrate addition if using fluorescence microscopy. Each apoptotic induction scenario may have different rates of Magic Red™ substrate hydrolysis and conversion to the red fluorescent product.

Call 1-800-829-3194 for technical assistance or email Technical Support: help {at} immunochemistry.com.