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DAPGreen - Autophagy Detection

Item # Unit Size
D676-10
5 nmol

For Research Use Only Products

∼ Features ∼
- Quantitative Analysis by Plate Reader
- High Correlation with Autophagy Marker LC3
- No Need for Transfection

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





Detection Principle
Simple Procedure: Just Add the Reagent
High Correlation with LC3
Quantitative Analysis by Flow Cytometer
Quantitative Analysis by Microplate Reader
DAPGreen Excitation/Emission
The Number of Usuable Assays
Usage examples from Publications
Related Product: DALGreen and DAPGreen
Quantification by fluorescence microscopy

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




The Number of Usable Assays


Microscopy: 250 μl /assay (8 well chamber slide)
Flow cytometry: 2,000 μl (6 well plate)
Plate reader: 100 μl/assay (96 well plate)

[Condition]
・Final concentration of DAPGreen working solution: 0.1 μmol/l
・The total volume prepared at 0.1 μmol/l DAPGreen working solution: 50 ml

NOTE: The number of assay depends on the final concentration of DAPGreen or volume of working solution.


Usage examples from Publications

Publications

1.Role of Glucosylceramide in Lung Endothelial Cell Fate and Emphysema
K. Koike et al., Am J Respir Crit Care Med., 2019, 200(9), 1113-1125. 

2.Augmenting Tumor-Starvation Therapy by Cancer Cell Autophagy Inhibition 
B. Yang et al., Adv.Sci., 2020, 7, 1902847. 

3.De Novo-Designed Near-Infrared Nanoaggregates for Super-Resolution Monitoring of Lysosomes in Cells, in Whole Organoids, and in Vivo 
H. Fang et al., ACS Nano., 2019, 13(12), 14426-14436. 


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.






Quantification by fluorescence microscopy

HeLa cells in a 96-well plate were stained with DAPGreen. Then, autophagy was induced through serum starvation before being detected via fluorescence microscopy.
The mean fluorescent intensity (MFI) per field of view was then quantified, using the Nikon software NIS-Elements. The results indicated that the MFI was increased in the serum-free HeLa cells.



<Detection conditions>
Ex = 488 nm, Em = 500-550 nm
Microscope Objective Lens: CFI Plan Apochromat VC 20x
Operation: Resonant Scanning
Lateral resolution (x-y): 512 x 512

<Equipment>
Confocal Microscope: Nikon A1R
Software: NIS-Elements

<Procedure>
1. Prepare HeLa cells for the assay in 96-well microplates and incubate at 37oC for overnight.
2. Wash once with culture medium (with serum) after removing the supernatant.
3. Add DAPGreen working solution and incubate at 37oC for 30 minutes.
4. Wash twice with culture medium (with serum) after removal of supernatant.
5. Add medium (with or without serum) to the wells where autophagy is to be induced and incubate at 37oC for 6 hours.
6.Fix HeLa cells with 4% PFA and observe under a confocal microscope.


Furthermore, the cells were observed in more detail by switching to a 100X objective. The number of DAPGreen fluorescent puncta per cell were then counted, and an average of 27 puncta were detected in HeLa cells cultured in serum-free medium, while only the 1.5 fluorescent puncta per cell were detected with serum. This was a very significant increase compared to cells in serum-containing medium.



<Detection conditions>
Ex = 488 nm, Em = 500-550 nm
Microscope Objective Lens: CFI Apochromat TIRF 100xC Oil
Operation: Galvano Scanning
Lateral resolution (x-y): 512 x 512

<Equipment>
Confocal Microscope: Nikon A1R
Software: NIS-Elements
※This data was provided by the user.


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.
2) L. Hu, T. Zhang, D. Liu, G. Guan, J. Huang, P. Proksch, X. Chen and W. Lin, "Notoamide-type alkaloid induced apoptosis and autophagy via a P38/JNK signaling pathway in hepatocellular carcinoma cells", RSC Adv., 2019, 9, 19855.
3) Q. Chu, S. Zhang, M. Chen, W. Han, R. Jia, W. Chen and X. Zheng, "Cherry Anthocyanins Regulate NAFLD by Promoting Autophagy Pathway", Oxid Med Cell Longev ., 2019,DOI:10.1155/2019/4825949.
4) K. Koike, E. V. Berdyshev, A. M. Mikosz, I. A. Bronova, A. S. Bronoff, J. P. Jung, E. L. Beatman, K. Ni, D. Cao, A. K. Scruggs, K. A. Serban and I. Petrache, "Role of Glucosylceramide in Lung Endothelial Cell Fate and Emphysema", Am. J. Respir. Crit. Care Med. ., 2019,DOI:10.1164/rccm.201812-2311OC.
5) F. Hongbao,Y. Shankun, C. Qixin, L. Chunyan, C. Yuqi, G. Shanshan, B. Yang, T. Zhiqi, L. Z. Amanda, T. Takanori, C.Yuncong, G. Zijian, H. Weijiang and D. Jiajie , "De Novo-Designed Near-Infrared Nanoaggregates for Super-Resolution Monitoring of Lysosomes in Cells, in Whole Organoids, and in Vivo.", ACS Nano, 2019, 13, (12), 1446.
6) B. Yang, L. Ding, Y. Chen and J. Shi, "Augmenting Tumor-Starvation Therapy by Cancer Cell Autophagy Inhibition", Adv. Sci., 2020,DOI:10.1002/advs.201902847.
7) Y. Tan, L. Yin, Z. Sun, S. Shao, W. Chen, X. Man,Y. Du and Y. Chen, "Astragalus polysaccharide exerts anti-Parkinson via activating the PI3K/AKT/mTOR pathway to increase cellular autophagy level in vitro.", Int. J. Biol. Macromol., 2020, DOI:10.1016/j.ijbiomac.2020.02.282.
8) Z. Peng, Y. Liao, X. Wang, L. Chen, L. Wang, C. Qin, Z. Wang, M. Cai, J. Hu, D. Li,P. Yao, A. K. Nüssler, L. Liu and W. Yang, "Heme oxygenase-1 regulates autophagy through carbon-oxygen to alleviate deoxynivalenol-induced hepatic damage", Arch. Toxicol., 2020, 94(2), 573.
9) J. Kim, W.Y.Chee, N. Yabuta, K. Kajiwara, S. Nada and M. Okada, "Atg5-mediated autophagy controls apoptosis/anoikis via p53/Rb pathway in naked mole-rat fibroblasts", Biochem. Biophys. Res. Commun., 2020, 22, DOI:10.1016/j.bbrc.2020.05.083.