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DAPGreen - Autophagy Detection

Item # Unit Size
D676-10
5 nmol

For Research Use Only Products



Content: 5 nmol x 1
Storage Condition :Store at -20 oC and protect from light
Shipping Condition :Ambient Temperature

Detection Principle

DAPGreen is used to detect autophagy in live cells. 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. DAPGreen, is a small fluorescent molecule, detects autophagosomes and autolysosomes possibly by a mechanism that the dye is incorporated into autophagosome during double membrane formation due to structure features, and then emits fluorescence under hydrophobic conditions. DALGreen is cell permeable, has no requirement of transfection method, and enables live cell imaging with fluorescence microscopy and quantitative assay by flow cytometry. For monitoring autolysosome, DALGreen [D675] is recommend since it allows detection of phagosome-lysosome fusion.



When an autophagosome membrane is formed, DAPGreen is incorporated inside of the membrane. The fluorescence of incorporated DAPGreen is enhanced under lipophilic condition. The analysis of DAPGreen also has a high correlation with that of LC3 which is a well-known autophagy marker. For details, please refer to the experimental data of DAPGreen.



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.




High Correlation with LC3


Result:
Almost all DAPGreen signals were colocalized with LC3.

Imaging Condition:
DAPGreen:Ex. 488 nm/Em. 500-563nm
Scale bar: 10 μm

The Condition of Autophagy Induction:
After adding DAPGreen to the RFP-LC3 expressed Hela cells, cells were treated with rapamycin to induce autophagy. Fluorescent imaging was conducted by confocal microscopy after 4 hrs. from autophagy induction.


Quantitative Analysis by Flow Cytometer

Result:
After 3 hrs. of incubation under starved condition, strong fluorescence was detected.

Detection:
Wavelengths: Ex. 488 nm / Em. 500-560 nm

The Condition of Autophagy Induction:
After staining with DAPGreen, HeLa cells were incubated for 0, 3, 6 hrs. with amino acid-free medium and detected by a flow cytometer.


Quantitative Analysis by Microplate Reader

Result:
After 2hrs. of incubation under starved condition fluorescence was observed. It was ca. 3,5 times stronger than "Control".

Detection:
Wavelengths: Ex. 450nm/ Em. 535nm

The Condition of Autophagy Induction:
After staining with DAPGreen, HeLa cells were incubated for 0, 2, 4, 6 hrs. with amino acid-free medium and detected by a microplate reader.


DAPGreen Excitation/Emission




Related Product Information

Detection is possible with a fluorescent microscope, a flow cytometer and a microplate reader using DAPGreen. DALGreen (D675) detects autolysosome. After a lysosome fuses with the autophagosome, the environment in the autolysosome become acidic. DALGreen fluoresce stronger as acidity increases. DALGreen can be applied in two methods (a fluorescent microscope and a flow cytometer). Please select your most suitable method depending on your equipment.






Reference
1) 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.