Magic Red™ Kits
Detect caspases or cathepsins in vitro and in real time
Magic Red kits allow researchers to monitor protease activity over time, offering a clear picture of protease-positive versus protease-negative cells. Just add MR to the media and analyze. As protease activity increases, positive cells will start to fluoresce.
These unique kits are not ELISAs and do not use antibodies - instead they use cell-permeant substrates specifically targeted by active enzymes. The target substrate peptide sequence is linked to a red fluorophore, Magic Red™ (also known as cresyl violet), which fluoresces once the substrate is cleaved by the specific enzyme (caspases 3 & 7, cathepsin B, cathepsin K, cathepsin L).
As protease activity progresses and more substrate is cleaved, the red fluorescent signal increases. Only cells with active enzymes will fluoresce, so you don’t get any signal from pro-enzymes or inactive forms of the enzyme. ICT’s Magic Red kits give a clear picture of protease-positive versus protease-negative cells. MR kits are true substrate assays - the red fluorescent label is quenched until it is actually cleaved by the active enzyme, so you can actually see the color develop over time.
Just culture your cells, add Magic Red to the media, incubate, and analyze. Once Magic Red is added to the media, it will pass through the cell membrane - no lysis or permeabilization steps are required. If it is cleaved by an active protease, the MR fluorophore will stay inside the cell, and often aggregates inside lysosomes. Cathepsin enzymes are lysosomal; caspases are not. Watch the cells through a microscope (and protect from light when not taking pictures) or read total fluorescence of the sample with a fluorescence plate reader (using a black microtiter plate).
Magic Red kits do not need a wash step. In fact, we discourage additional manipulation of the cells as the Magic Red fluorophore may migrate back out of the cell. Adherent cells can be trypsinized and transferred into a black microtiter plate for analysis with a fluorescence plate reader. We have successfully used these kits with Jurkat, HL-60, THP-1, fibroblasts, UMUC-3, MCF-7, and U937 cells. Protect the cells from light as the reagent will photobleach over time.
Magic Red kits work well with cells that cannot be centrifuged, with adherent cells, with thin frozen sections, and can be adapted for hi-throughput screening. You can use FLICA™ and Magic Red together for dual-staining studies. Magic Red excites at 540-590 and emits at >610nm. This signal can be detected with a fluorescence microscope, a fluorescence plate reader, or special flow cytometers with adjustable excitation wavelengths.
Rat fibroblasts were seeded in 12 well plates at 10,000 cells in 1mL and irradiated the following day. 500uL was removed; 23.1uL of MR-DEVD solution (cat. #935) was mixed with 200uL media and added. 1 hour later, 300uL media was added and cells were photographed over 16 hours. Bright field image shows increasing red intensity as caspase activity and apoptosis progressed. Time 0 is at left, 16 hours is at right. Data courtesy of Dr. Martin Purschke, MA General Hospital. Watch the color increase in the movie clip of these cells in a bright field.
ICT's MR Caspases 3&7 kit clearly distinguishes apoptotic MCF-7 cells from non-apoptotic cells. Apoptotic cells exhibit red lysosomes (MR) with less intense blue nuclei. These cells are intermixed with non-apoptotic cells bearing bright blue nuclei (Hoechst) and absent lysosomal staining.
Sample protocol:
- Culture your cells individually or up to 1 x 10^6 cells/mL.
- Induce your experimental conditions following your protocol.
- Reconstitute the reagent with DMSO to form the stock concentrate (which can be frozen for future use).
- Dilute the stock concentrate with 1X PBS to form the working solution.
- Add ~10mcL of the working solution directly to a 300-500mcL aliquot of your cell culture for labeling.
- Incubate, typically 1-4 hours; protect cells from light.
- If desired, label DNA with Hoechst stain.
- If desired, label lysosomes with Acridine Orange.
- If desired, fix cells.
- Analyze data using a fluorescence microscope, or plate reader.