DALGreen 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. DALGreen, which is a small hydrophobic molecule, passes the plasma membrane of live cells and is incorporated in the autophagosome. After a lysosome fuses with the autophagosome, the environment in the autolysosome become acidic. DALGreen fluoresce stronger as acidity increases. The quality of this dye enables live cell imaging with a fluorescence microscopy and quantitative assay by flow cytometry.
When an autophagosome membrane is formed, DALGreen is incorporated inside of the autophagosome membrane when the membrane is formed. The fluorescence of DALGreen is enhanced under acidic condition after the autophagosome is fused with the lysosome.
First, cells are plated for the assay. After discarding the supernatant and washing the cells with culture medium, DALGreen working solution is added. While incubating for 30 minutes, DALGreen passes the plasma membrane of live cells and is incorporated in the forming autophagosome (Fig.1). After the 30 minutes incubation, supernatant is removed and the cells are washed. Medium containing autophagy-inducing agent is added to the cells. After incubating, observation can be made via fluorescence microscope or flow cytometer. For more detailed procedure, please refer to the manual.
Procedure Comparison between DALGreen and Existing Reagents
Comparison to LC3
Method of autophagy induction
Control: incubated in the cell media for 6 hours
Starved: incubated in media without amino acid for 6 hours
Condition of DALGreen Imaging
Cell line: HeLa
Wavelength: Ex. 488 nm/ Em. 500-563 nm
Scale bar: 20 μm
Comparison to MDC
DALGreen can be used for live autophagy monitoring because the Working Solution is added prior to inducing autophagy unlike MDC.
Time-lapse imaging of autophagy
For more details, click application data
The Number of Usable Assay
Microscopy: 250 μl /assay (8 well chamber slide)
Flow cytometry: 500 μl (24 well plate)
・The total volume prepared at 1.0 μmol/l DALGreen working solution: 20 ml
NOTE: The number of assay depends on the final concentration of DALGreen or volume of working solution.
Usage example of publications
LC3 is one of markers that is used in autophagy detection1). However, whether or not autophagy is induced can’t be determined from only elevation of autophagy markers such as LC32). To increase the reliability of data, multiple assay approach is used in combination with western blot or different assay3).
Dojindo’s DALGreen, DAPGreen and DALRed can be used for the detection of fluorescent imaging. Their probes allows to visualize autophagosome/autolysosome selectively. DALGreen is used for autolysosome detection with western blot of LC3 4), 5).
1) I. Tanida, et al., “LC3 and Autophagy.” Methods Mol Biol., 2008, 445, 77-88.
2) DJ. Klionsky, et al., “Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition).”, Autophagy, 2016, 12(1), 1-222
3) S. Barth, et al., “Autophagy: assays and artifacts”, 2010, 221(2): 117–124.
4) J. Xia, et al., “NEK2 Induces Autophagy-Mediated Bortezomib Resistance by Stabilizing Beclin-1 in Multiple Myeloma”, Mol Oncol., 2020, 14(4), 763-778.
5) L. Cui, et al., “The Lysosomal Membrane Protein Lamp2 Alleviates Lysosomal Cell Death by Promoting Autophagic Flux in Ischemic Cardiomyocytes”, Front. Cell Dev. Biol., 2020, 8, 31.
6) H. Fang et al., “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), 14426-14436.
|Fluorescent microscope||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 Letters., 2018, 592, (4), 559–567.|
|Fluorescent microscope||T. Sakata, A. Saito and H. Sugimoto, “In situ measurement of autophagy under nutrient starvation based on interfacial pH sensing.”, Scientific Reports., 2018, 8, 8282.|
|Fluorescent microscope||Y. Egawa, C. Saigo, Y. Kito, T. Moriki and T. Takeuchi , “Therapeutic potential of CPI-613 for targeting tumorous mitochondrial energy metabolism and inhibiting autophagy in clear cell sarcoma.”, PLoS One., 2018, 13, (6), e0198940.|
|Fluorescent microscope||S. Abe, S. Hirose, M. Nishitani, I. Yoshida, M. Tsukayama, A. Tsuji and K. Yuasa , “Citrus peel polymethoxyflavones, sudachitin and nobiletin, induce distinct cellular responses in human keratinocyte HaCaT cells.”, Biosci. Biotechnol. Biochem. ., 2018, 82, (12), 1347.|
(Quantified using analysis software)
|W. Yuping, M. Congshun, Z. Huihui, Z. Yuxia, C. Zhenguo and W. Liping, “Alleviation of endoplasmic reticulum stress protects against cisplatin-induced ovarian damage.”, Reprod. Biol. Endocrinol., 2018,doi: 10.1186/s12958-018-0404-4.|
|S. Xue, F. Mao, D. Hu, H. Yan, J. Lei, E. Obeng, Y. Zhou, Y. Quan, and W. Yu, “Acetylation of BmAtg8 inhibits starvation-induced autophagy initiation.”, Mol. Cell Biochem., 2019,doi: 10.1007/s11010-019-03513-y.|
|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.|
|FCM||E. Sasabe, A. Tomomura, N. Kitamura and T. Yamamoto, “Metal nanoparticles-induced activation of NLRP3 inflammasome in human oral keratinocytes is a possible mechanism of oral lichenoid lesions.”, Toxicol In Vitro., 2020, 62, 104663.|
|Fluorescent microscope||J. Xia, Y. He, B. Meng, S. Chen, J. Zhang, X. Wu, Y. Zhu, Y. Shen, X. Feng, Y. Guan, C. Kuang, J. Guo, Q. Lei, Y. Wu, G. An, G. Li, L. Qiu, F. Zhan and W. Zhou, “NEK2 induces autophagy-mediated bortezomib resistance by stabilizing Beclin-1 in multiple myeloma.”, Mol Oncol, 2020, DOI: 10.1002/1878-0261.12641.|
|Fluorescent microscope||Q. Xu, W. Shi, P. Lv, W. Meng, G. Mao, C. Gong, Y. Chen, Y. Wei, X. He, J. Zhao, H. Han, M. Sun and K. Xiao, “Critical role of caveolin-1 in aflatoxin B1-induced hepatotoxicity via the regulation of oxidation and autophagy.”, Cell Death Dis., 2020, 11(1), 6.|
(Cardiac muscle cells)
|Fluorescent microscope||L Cui, LP Zhao, JY Ye, L Yang, Y Huang, X.P. Jiang, Q. Zhang, JZ. Jia, DX. Zhang and Y. Huang, “The Lysosomal Membrane Protein Lamp2 Alleviates Lysosomal Cell Death by Promoting Autophagic Flux in Ischemic Cardiomyocytes.”, Front Cell Dev Biol, 2020,DOI:10.3389/fcell.2020.00031.|
|Fluorescent microscope||Y Yang, J Huang, J Li, H Yang and Y. Yin, “The Effects of Butyric Acid on the Differentiation, Proliferation, Apoptosis, and Autophagy of IPEC-J2 Cells..”, Curr. Mol. Med., 2020, 20(4), 307.|
(fibroblast, renal epithelial cell)
|Fluorescent microscope||M. M. Ivanova, J. Dao, N. Kasaci, B. Adewale, J. Fikry and O. G. Alpan , “Rapid Clathrin-Mediated Uptake of Recombinant α-Gal-A to Lysosome Activates Autophagy”, Biomolecules , 2020, 10(6), 837.|
|Fluorescent microscope||S. Ikeoka and A. Kiso , “The Involvement of Mitophagy in the Prevention of UV-B-Induced Damage in Human Epidermal Keratinocytes “, J. Soc. Cosmet. Chem. Jpn., 2020, 54(3), 252.|