Autophagy : Reagent Selection Guide

Science Note

New Mitophagy Pathways: Next Therapy Targets? [Sep. 23, 2025] 

Defects in mitophagy through the autophagy–lysosome pathway, particularly in the PINK1/Parkin system, are closely linked to neurodegenerative disease. Recent studies reveal additional mechanisms, including a mitophagic stress response that relieves autophagy inhibition and a PINK1–SMAD3 transcriptional loop, highlighting new therapeutic targets in mitochondrial quality control.

1. Mitochondrial damage triggers the concerted degradation of negative regulators of neuronal autophagy (Nature Communications, 2025)
Summary: Damaged mitochondria are usually cleared by the Pink1/Parkin pathway, but this study identifies a parallel Mitophagic Stress Response (MitoSR) triggered by mitochondrial damage in neurons. MitoSR degrades autophagy inhibitors (MTMR5, MTMR2, Rubicon) via the ubiquitin–proteasome system, thereby enhancing autophagosome formation, lysosome fusion, and overall mitochondrial quality control.

Highlighted technique: The study used fluorescent pH probes to monitor lysosomal acidity under mitochondrial stress. With this approach, the authors showed that reducing Rubicon, a protein that suppresses autophagy, restored lysosomal activity and enabled more efficient clearance of damaged mitochondria.

 Related techniques  Lysosomal Function Analysis (as used in this article), Autophagosome Detection (as used in this article)

2. A positive feedback loop between SMAD3 and PINK1 in regulation of mitophagy (Cell Discovery, 2025)
Summary: PINK1 is a protein that protects cells by helping remove damaged mitochondria, and SMAD3 is a transcription factor that controls gene activity. This study reveals, for the first time, that PINK1 and SMAD3 activate each other in a unique self-amplifying loop independent of the usual TGFβ pathway, uncovering a powerful new mechanism that strengthens mitochondrial quality control and offers fresh insight into diseases like Parkinson’s.

Highlighted technique: To evaluate mitophagy, researchers used the Mito-Keima assay, which tracks mitochondria entering acidic lysosomes as a measure of degradation. In HeLa cells expressing YFP-Parkin and mito-Keima, SMAD3 knockdown combined with Oligomycin/Antimycin A treatment enabled flow cytometric quantification of this process.

 Related techniques  Mitophagy DetectionAutophagic Flux Assay Kit

Previous Science Note

Related Techniques (click to open/close)
Target Kits & Probes
Mitophagy  detection Mitophagy Detection Kit
First-time autophagy research Autophagic Flux Assay Kit
Autophagy detection DAPGreenDAPRed (Autophagosome detection), DALGreen (Autolysosome detection)
Lysosomal function Analysis Lysosomal Acidic pH Detection Kit -Green/Red and Green/Deep Red
Mitochondrial membrane potential detection JC-1 MitoMP Detection Kit, MT-1 MitoMP Detection Kit
Mitochondrial superoxide detection MitoBright ROS Deep Red - Mitochondrial Superoxide Detection
ROS Detection ROS Assay Kit -Highly Sensitive DCFH-DA- and ROS Assay Kit -Photo-oxidation Resistant DCFH-DA-
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)
  Accurate Autophagy Imaging with Lysosome Staining

We performed fluorescence imaging under amino acid starvation in HeLa cells stained with the autophagosome detection probe DAPRed (Code: D677) and LysoPrime Green (Code: L261). The fluorescence signal of LysoPrime Green increased, and lysosomal localization was confirmed over time. These results indicate that the co-localization rate with DAPRed fluorescence is sufficient, enabling accurate autophagy analysis.

 Application Note II  (click to open/close)
  Induction of Mitophagy in Parkin Expressed HeLa cells

CCCP(carbonyl cyanide m-chlorophenyl hydrazone) has been added to normal and Parkin expressed cells. The strong fluorescence was not observed in normal HeLa cells(A)(B). On the other hand, the trong fluorescence was shown in Parkin expressed cells in 18 hours after additon of CCCP(C). Some of the puncta are co-localized with the autophagy marker(GFP-LC3). In addition, suppressed fluorescence of Mtphagy dye was observed when autophagy inhibitor, bafilomycin was added to Parking expressed cells(D). Because lysosomal pH was increased by the additon of bafilomycin.

Previous Science Note  

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!
      
(Click to open)

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 closes 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 property

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.

 

 


 


 

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