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.
- Prepare samples and controls
- Reconstitute Magic Red with 100 or 400 µL DMSO
- Dilute Magic Red 1:5 with diH2O
- Add diluted Magic Red at 1:15-1:30 to suspension cells
- Incubate while protected from light.
- Watch color start to develop within 15 minutes of addition of Magic Red
- If desired, label with additional stains, such as Hoechst, DAPI, or an antibody.
- 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.
Product Specific References
|Benavides, R.A.S., et al. 2023. The HL-60 human promyelocytic cell line constitutes an effective in vitro model for evaluating toxicity, oxidative stress and necrosis/apoptosis after exposure to black carbon particles and 2.45 GHz radio frequency. The Science of the total environment, 161475.
|Tong, J., et al. 2023. Measuring Caspase Activity Using a Fluorometric Assay or Flow Cytometry. Journal of visualized experiments : JoVE.
|Procházková, M., et al. 2023. Novel Förster Resonance Energy Transfer probe with quantum dot for a long-time imaging of active caspases inside individual cells. Analytica chimica acta, 341334.
|Ndinyanka Fabrice, T., et al. 2022. An evolutionarily conserved coronin-dependent pathway defines cell population size. Science signaling, eabo5363.
Question: The customer wish to combine in the same sample the Magic Red staining and Annexin V staining. The analysis of the samples will be performed on a flow cytometer. In the manual, page 10, section: single satin Flowcytometry, after the MR incubation there is no wash step. Since the Annexin V staining procedure can be performed only with a staining buffercontaining calcium, can the MR be performed in this buffer instead of the cells media? So that the customer will begin with the MR staining and in the last 15 min she will add the Annexin V. Another option can be perhaps to stain with the MR, wash and replace it to the Annexin V buffer for future 15 min staining. Will the MR “survive” a centrifuge?
Answer: We have not evaluated our Magic Red substrates in combination with Annexin V, so unfortunately can’t say with certainty that this will work. However, that said, we would recommend against the proposed sequence where the customer stains with MR, washes, resuspends in Annexin V buffer, and then completes the Annexin V staining step. MR substrate is not covalently coupled in the cell and the signal would be washed away in the centrifugation step. The customer can try completing Annexin V staining and then carrying our MR staining afterwards in the Annexin V buffer. We would once again caution that the use of this technique will need to be evaluated experimentally. We recommend including positive and negative single stain controls for interpretation of the results.