Autophagy : Reagent Selection Guide

Why is Autophagy research important?

Autophagy is essential in maintaining cellular homeostasis by clearing damaged proteins and organelles. In cancer, it can act both as a tumor suppressor and promoter, making it a key therapeutic target. For neurodegenerative diseases such as Alzheimer’s and Parkinson’s, autophagy helps prevent toxic protein accumulation, thus potentially delaying disease progression. Age-related decline in autophagy contributes significantly to cellular dysfunction and aging. Therefore, modulating autophagy offers promising therapeutic strategies for cancer, neurodegenerative disorders, and aging-related conditions.   Master the Basics with a Overview Map!
      
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Science Note

Autophagy-Glycolysis Crosstalk Maintains Cellular Homeostasis [Apr. 22, 2025] 

Autophagy and glycolysis are fundamental to maintaining cellular energy homeostasis, yet their imbalance can lead to dysfunction and disease. Recent studies reveal how impaired autophagy disrupts glucose metabolism and how excessive glycolytic flux can suppress autophagy.This Science Note introduces new insights into the dynamic crosstalk between these pathways and its impact on cellular homeostasis.

Autophagy regulator ATG5 preserves cerebellar function by safeguarding its glycolytic activity (Nature Metabolism, 2025)
In cerebellar Purkinje cells involved in motor control, autophagy regulates glucose uptake by limiting the amount of GLUT2. Loss of autophagy leads to excessive glucose uptake and dysregulation of glycolysis, resulting in the accumulation of by-products such as lysophosphatidic acid and serine, which contribute to neuronal degeneration.

Highlighted technique: To assess changes in glucose uptake in autophagy-deficient Purkinje cells, the authors used cerebellar organotypic slice cultures from mice and evaluated glucose uptake capacity by fluorescent microscopy using a fluorescent analog of glucose.

 Related technique  Autophagic Flux Assay, Highly Sensitive Fluorescent Glucose Analog

Weak neuronal glycolysis sustains cognition and organismal fitness (Nature Metabolism, 2024)
Neurons maintain low glycolytic activity to maintain NAD⁺ levels, which are essential for sirtuin-mediated autophagy and mitochondrial homeostasis. Aberrant activation of glycolysis depletes NAD⁺, suppresses autophagy and leads to mitochondrial dysfunction and cognitive decline.

Highlighted technique: In this study, a mouse model was generated by overexpressing PFKFB3, a glycolysis-promoting enzyme that is normally degraded in neurons, to investigate the importance of weak neuronal glycolysis. Tissues and cells derived from these mice were used to examine NAD⁺ levels, mitochondrial activity, and autophagy.

 Related technique  NAD/NADH Assay, Glycolysis/OXPHOS AssayMitochondrial ROS Detection 

Autophagy counters inflammation-driven glycolytic impairment in aging hematopoietic stem cells (Cell Stem Cell, 2024)
Age-related inflammation impairs glucose uptake in HSCs, causing metabolic stress and functional decline, while this stress triggers adaptive autophagy in a subset of HSCs that supports functional maintenance. Consistent with this mechanism, transient induction of autophagy by short-term fasting restores metabolism and revives the regenerative capacity of aged HSCs.

Highlighted technique: In this study, HSCs were isolated from aged mice subjected to a fasting/refeeding regimen and transplanted into recipient mice to assess their regenerative capacity. FCM was used to detect donor-specific markers and quantify the expansion of donor-derived cells, allowing evaluation of the functional restoration of HSCs by the fasting/refeeding treatment.

 Related technique  Autophagic Flux Assay, Extracellular OCR AssayGlucose Uptake Assay

Previous Science Note

Related Techniques (click to open/close)
Target Kit & Probes
First-time autophagy research Autophagic Flux Assay Kit
Autophagy detection DAPRed (Autophagosome detection), DALGreen (Autolysosome detection)
Mitophagy  detection Mitophagy Detection Kit
Highly Sensitive Fluorescent Glucose Analog Glucose Uptake Assay Kit Blue/ Green / Red
Glycolysis/Oxidative phosphorylation Assay Glycolysis/OXPHOS Assay Kit
Mitochondrial superoxide detection MitoBright ROS Deep Red - Mitochondrial Superoxide Detection
Oxygen consumption rate assay Extracellular OCR Plate Assay Kit
Mitochondrial membrane potential detection JC-1MitoMPDetection Kit, MT-1MitoMPDetection Kit
Apoptosis detection in multiple samples Annexin V Apoptosis Plate Assay Kit
Cell proliferation/ cytotoxicity assay Cell Counting Kit-8 and Cytotoxicity LDH Assay Kit-WST
 Application Note I  (click to open/close)
  NAD+ Depletion and Autophagy-Lysosomal Pathway Response

We determined how FK866-induced lysosomal deacidification affects the autophagy-lysosomal pathway. After staining with DAPGreen/DAPRed (for detecting autophagosome), or DALGreen (for detecting autolysosome), HeLa cells were starved in HBSS incubation and then treated with FK866 or Bafilomycin A1. Under the starvation condition, the fluorescent signals from all dyes increased, indicating the proceeding autophagy-lysosomal pathway. On the other hand, only DALGreen's signals were decreased in FK866 and Bafilomycin A1 treated cells with starvation conditions. These results clearly suggested that FK866 inhibits the autophagy-lysosomal pathway by NAD+ depletion-induced lysosomal deacidification.

Nampt inhibitor, FK866 inhibits the progress of autophagosome to autolysosome by lysosomal deacidification. A recent finding shows that the dysfunctional condition of nicotinamide adenine dinucleotide (NAD+) biosynthetic enzyme, Nampt induces lysosomal deacidification1). In this section, we tried to determine how NAD+ depletion-induced lysosomal deacidification affects the autophagy-lysosomal pathway.  1) Mikako Yagi, et. al., EMBO J., 40(8), e105268 (2021)

 Application Note II  (click to open/close)
  Simultaneous Detection of Lysosomal and Mitochondrial Dysfunction

We tried the simultaneous detection of lysosomal and mitochondrial dysfunction in Hela cells treated with CCCP or Antimycin (AN). CCCP and AN are well-known inducers of mitochondrial ROS regarding loss of mitochondrial membrane potential. Recent research showed the result that CCCP induces not only mitochondrial ROS but also lysosomal neutralization. To detect mitochondrial ROS, HeLa cells were labeled by MitoBright ROS Deep Red - Mitochondrial Superoxide Detection, and the lysosomal mass and pH were detected separately with LysoPrime Green and pHLys Red. Co-staining with MitoBright ROS and Lysosomal dyes demonstrated that CCCP causes lysosomal neutralization and mitochondrial ROS induction at the same time.

Products in Use
   - Lysosomal Acidic pH Detection Kit
     (combination kit of LysoPrime Green / pHLys Red)
   - MitoBright ROS Deep Red - Mitochondrial Superoxide Detection

 

 

Previous Science Note  

  

What is Autophagy?

Autophagy is a degradation process of cytoplasmic dysfunctional proteins and organelles. In this process, an isolation membrane composed of a double membrane appears in the cytosol, gradually expands, encloses the aggregated proteins and damaged organelles, and close to form autophagosomes. The autophagosomes are fused with lysosomes to form autolysosomes, which have an acidic environment. The contents in autolysosomes are decomposed by digestive enzymes in lysosomes. Since this cellular function is said to be related to aging as well as neurodegenerative diseases such as Parkinson’s disease, a simple autophagy detection method is being required.

 

Autophagy Reagents Selection Guide

Depending on the method and purpose of autophagy assessment, three types of fluorescent small molecule reagents and kits are available.

Select by targets

  Small Fluorescent Molecules Fluorescent Protein
DAPGreen  DAPRed  DALGreen Autophagic Flux Assay Kit GFP-LC3 RFP-LC3 mRFP-GFP-LC3
Autophagosome - Autophagosomes and autolysosomes are each detectable. Autophagosomes and autolysosomes are each detectable.
Autolysosome -
Transfection No need for transfection

Autophagic Flux Assay KitThis kit contains autophagosome and autolysosome detection dye (DAPRed), autolysosome detection dye (DALGreen), and lysosomal acidification inhibitor (bafilomycin A1). The Autophagic Flux Assay Kit allows the accurate evaluation of autophagic flux by monitoring autophagosome formation, lysosome fusion, and digestion of contents.


Select by detector and fluorescence propety

There are several autophagy-related products available, which one to choose?

Example: Screen the autophagy activity of a drug

The first step is to select candidates from multiple samples using a plate reader with DAPGreen.

Once the change in autophagic activity has been detected, imaging with Autophagic Flux Assay Kit to confirm whether autophagy is activated or inhibited.

 

Principle of Autophagy Reagents

DALGreen / DAPGreen is incorporated into hydrophobic lipid bilayers due to its similar structure to membrane phospholipids such as phosphatidylethanolamine.

DALGreen and DAPGreen were each incorporated during liposome membrane formation and observed by confocal microscopy; DAPGreen showed strong fluorescence upon incorporation into the lipid bilayer, whereas weak fluorescence was observed for DALGreen. Scale bar: 20 μm


DAPGreen: fluorescence intensity increases in response to the hydrophobic field environment within the lipid bilayer (detects both autophagosomes and autolysosomes)



DALGreen : incorporated into lipid bilayers, fluorescence intensity increases in acidic environment (detects autolysosomes)

For detailed experimental results, including the specificity of autophagy, see the original paper here.

DAPGreen/DALGreen: H. Iwashita, et al., "Small fluorescent molecules for monitoring autophagic flux", FEBS Letters., 2018592, (4), 559–567.
DAPRed:H. Sakurai, et al"Development of small fluorescent probes for the analysis of autophagy kineticsiScience​, 202326, 107218.
 

Typical Examples of Article in Use

Title Discovery and Structure-Based Optimization of Novel Atg4B Inhibitors for the Treatment of Castration-Resistant Prostate Cancer
Kudo, Y. et al., Journal of Medical Chemistry2022, 65(6)
Purpose Evaluation of ATG4B inhibitors using autophagy activity as an indicator.

Product/
Method

DAPGreen/Microscope

 

Title S-Nitrosylation of p62 Inhibits Autophagic Flux to Promote α-Synuclein Secretion and Spread in Parkinson's Disease and Lewy Body Dementia
Oh, C. et al., Journal of Neuroscience2022, 41(14), 3011-3024
Purpose Confirmation that p62(C331A) knock-in mutant causes autophagy inhibition (decrease in autolysosomes).
Product/
Method

DALGreen&DAPRed (Autophagic Flux Assay Kit)/Microscope

 

Title Scd1 and monounsaturated lipids are required for autophagy and survival of adipocytes
Mori, H. et al., Molecular Metabolism2024, 83, 101916
Purpose Confirmation that SCD1KO causes autophagy inhibition (decrease in autolysosomes).
Product/
Method

DALGreen&DAPRed (Autophagic Flux Assay Kit)/Microscope

 

 

Experimental Example: Analysis of autolysosome formation inhibition using Bafilomycin A1 by confocal fluorescence microscopy

DALGreen and DAPRed labeled HeLa cells were used to evaluate changes in autophagic flux induced by the lysosomal acidification inhibitor bafilomycin A1 (Baf. A1). Compared to starvation conditions, the fluorescence signals of DALGreen were decreased under inhibited conditions of autolysosome formation by the addition of Baf. A1. In contrast, the fluorescence signals of DAPRed were increased under the same conditions, indicating that Baf. A1 led to the accumulation of autophagosome.
 

 

<Experimental Conditions>

CTRL: Normal condition, Stv.: Induction of autophagy, Stv. + Baf. A1: Inhibition of autolysosome formation
DALGreen filter set: 488 nm (Ex), 490–550 nm (Em)
DAPRed filter set: 561 nm (Ex), 565–700 nm (Em)

 

<Procedure>

1. HeLa cells were seeded (1.0 x 104 cells/well) on a μ-slide 8 well plate (ibidi) and cultured overnight at 37°C in an incubator equilibrated with 95% air and 5% CO2.
2. After washing twice with MEM containing 10% fetal bovine serum, 200 μl of DALGreen/DAPRed working solution (DALGreen: 1 µmol/l, DAPRed: 0.2 µmol/l) and the cells were incubated at 37°C for 30 minutes.
3. The supernatant was discarded, and the cells were washed twice with MEM containing 10% fetal bovine serum.
4. Samples were prepared under the following conditions.

• MEM containing 10% fetal bovine serum (200 µl) was added to the well, and the cells were incubated at 37 °C for 2 hours 20 minutes. (Control)
• Amino acid-free medium (FUJIFILM Wako Pure Chemical Industries, Ltd., Catalogue code: 048-33575) (200 μl) was added to the well, and the cells were incubated at 37°C for 2 hours 20 minutes. (Starvation)
• Amino acid-free medium (200 μl) was added to the well, and the cells were incubated at 37°C for 2 hours. The supernatant was discarded, bafilomycin A1 working solution (10,000 times dilution, 200 μl), an inhibitor of lysosomal acidification, was added to the well, and the cells were incubated at 37°C for 20 minutes. (Inhibition of autolysosome formation)

5. The stained cells were observed under a confocal fluorescence microscope.

 

Experimental Example: Analysis of lysosomal protein degradation inhibition using E64d/Pepstatin A  by confocal fluorescence microscopy

DALGreen and DAPRed labeled HeLa cells were used to evaluate changes in autophagic flux induced by the inhibitor of lysosome enzymes E64d/Pepstatin A (Pep A). Compared to starvation conditions, the fluorescence signals of DALGreen and DAPRed were increased due to autolysosome accumulation by the addition of E64d/Pep A.

 

<Experimental Conditions>

CTRL: Normal condition, Stv.: Induction of autophagy, Stv. + E64d/Pep A: Inhibition of lysosomal protein degradation
DALGreen filter set: 488 nm (Ex), 490–550 nm (Em)
DAPRed filter set: 561 nm (Ex), 565–700 nm (Em)

 

<Procedure>

1. HeLa cells were seeded (1.0 x 104 cells/well) on a μ-slide 8 well plate (ibidi) and cultured overnight at 37 °C in an incubator equilibrated with 95% air and 5% CO2.
2. After washing twice with MEM containing 10% fetal bovine serum, 200 μl of DALGreen/DAPRed working solution (DALGreen: 1 µmol/l, DAPRed: 0.2 µmol/l) and the cells were incubated at 37°C for 30 minutes.
3. The supernatant was discarded, and the cells were washed twice with MEM containing 10% fetal bovine serum.
4. Samples were prepared under the following conditions.

• MEM containing 10% fetal bovine serum (200 µl) was added to the well, and the cells were incubated at 37°C for 2 hours. (Control)
• Amino acid-free medium (FUJIFILM Wako Pure Chemical Industries, Ltd., Catalogue code: 048-33575) (200 μl) was added to the well, and the cells were incubated at 37°C for 2 hours. (Starvation)
• Amino acid-free medium containing E64d/Pep A (10 µg/ml each, 200 μl), an inhibitor of lysosomal protease, was added to the well, and the cells were incubated at 37°C for 2 hours. (Inhibition of lysosomal protein degradation)

5. The stained cells were observed under a confocal fluorescence microscope.

 

 


 


 

Product Classification

Product Classification