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 and 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.

To use Magic Red, add the substrate directly to the cell culture media, incubate, and analyze. Because it is cell-permeant, it easily penetrates the cell membrane and the membranes of the internal cellular organelles - no lysis nor 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; caspases are not lysosomal enzymes. As protease activity progresses and more substrate is cleaved, the red fluorescent signal potential will intensify, enabling researchers to watch it increase over time and quantify apoptosis.

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.