Autophagy is an intracellular degradation system, where dysfunctional proteins and organelles are degraded. In this process, aggregated dysfunctional proteins are surrounded by the double membrane to form an autophagosome.
The small fluorescent molecule DAPRed is used to be detect autophagosomes and autolysosomes. The mechanism has been suggested to be that the dye is incorporated into the autophagosome during double-membrane formation via structural features, and then emits fluorescence under hydrophobic conditions. The utility of DAPRed is conferred by its molecular properties: it is permeable to cells, has no requirement for transfection, and enables live cell imaging with fluorescence microscopy. For monitoring autolysosomes, DALGreen (D675) is recommend because it enables the detection of phagosome-lysosome fusion.
Simple Procedure: Just Add the Reagent
Gene transfection is not necessary. You only need to add the reagent to your cell sample, and you can get a fluorescent image.
Co-staining of DAPRed and DALGreen
HeLa cells were stained with DAPRed, which is a dye-stained autophagosome, and DALGreen, which is a dye-stained autolysosome. Autophagy was then induced by starvation.
The fluorescence of DAPRed and DALGreen was stronger in starved HeLa cell culture.
DAPRed: Ex. 561 nm/Em. 600-700 nm
DALGreen: Ex. 488 nm/Em. 500-563 nm
Scale bar: 20 μm
After staining with DAPred and DALGreen, HeLa cell was incubated with culture medium or without amino acid-free medium for 5 hours.
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H. Iwashita, H. T. Sakurai, N. Nagahora, M. Ishiyama, K. Shioji, K. Sasamoto, K. Okuma, S. Shimizu, and Y. Ueno, “Small fluorescent molecules for monitoring autophagic flux”, FEBS Lett., 2018, 592, (4), 559–567