When are caspases activated?
ICTs Magic Red™ kits enable you to monitor protease activity over time and will give you 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, K, or 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 MR kits give you 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. Watch 2 movies of real-time caspase activity, courtesy of Dr. M. Purschke, MA General Hospital: movie clip of live cells (bright field) ; movie of red fluorescence only.
Just culture your cells, add MR to the media, incubate, and analyze. Once MR 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).
ICTs Magic Red™ kits do not need a wash step. In fact, we discourage additional manipulation of the cells as the MR 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.
MR 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 MR together for dual-staining studies. MR 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.
Magic Red™ Real-Time Protease Detection Kits
1. Culture your cells individually or up to 1 x 106 cells/mL.
2. Induce your experimental conditions following your protocol.
3. Reconstitute the reagent with DMSO to form the stock concentrate (which can be frozen for future use).
4. Dilute the stock concentrate with 1X PBS to form the working solution.
5. Add ~10uL of the working solution directly to a 300-500uL aliquot of your cell culture for labeling.
6. Incubate, typically 1-4 hours; protect cells from light.
7. If desired, label DNA with Hoechst stain.
8. If desired, label lysosomes with Acridine Orange.
9. If desired, fix cells.
10. Analyze data using a fluorescence microscope, or plate reader.
Lee, B., G. L. Johnson, S. A. Hed, Z. Darzynkiewicz, J. Talhouk, and S. Mehrotra. 2001. DEVDase detection in intact apoptotic cells using the cell permeant fluorogenic substrate, (z-DEVD)2-cresyl violet. Biotechniques vol 35 no 5:1080-1085. Read this publication.
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