Lysosome Function Analysis - Selection Guide for Detection and Imaging Reagent / Probe / Kit DOJINDO LABORATORIES

Why is Lysosomal Function Important?

Lysosome has been revealed to be an important organelle with a complex role in nutrient sensing and multifaceted signaling. Its importance has been emphasized in research fields as diverse as a neurological disease, cancer, immunity, and senescence. Recent research reveals that lysosome acidification declines in neurons well before extracellular amyloid deposition, thus lysosomal function is now a hot topic in Alzheimer's disease research.

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Science Note

[Aug. 27, 2025] Previous Science Note

New Lysosome Restoration Pathways for Disease and Aging

Discovery of New Transcriptional Programs Restoring Lysosomes Suggests New Therapeutic Opportunities
Lysosomes are essential organelles for maintaining cellular homeostasis, and their dysfunction is closely associated with a variety of diseases, including neurodegeneration, making it critical to understand how cells restore lysosomal function. Recent work in C. elegans showed that the GATA transcription factor ELT-2 activates a lysosomal surveillance response (LySR), thereby enhancing lysosomal activity and extending lifespan¹. In addition, studies in mouse models of lysosomal storage disorders identified STING, best known as a DNA sensor in innate immunity, as a lysosomal repair sensor that activates TFEB, the master regulator of lysosomal and autophagy genes, uncovering new pathways of lysosomal quality control and suggesting strategies to counter proteotoxicity and neurodegenerative disease².

1. A lysosomal surveillance response to stress extends healthspan (Nature Cell Biology, 2025)

Summary: Researchers found that silencing vacuolar H+-ATPase subunits in the intestine of C. elegans creates a stress that activates the lysosomal surveillance response (LySR), governed by the GATA transcription factor ELT-2, to boost lysosomal activity and clear protein aggregates. This mechanism enhances lysosomal function, extends lifespan, and suggests that activating the newly identified LySR pathway could be a strategy to treat lysosome-related diseases and promote healthy aging.

Highlighted technique: To examine how silencing v-ATPase subunits affects lysosomal function, the authors used two approaches in living C. elegans. They applied a fluorescent probe that labels lysosomes and a pH-sensitive lysosomal reporter to monitor changes in acidification, and these assays demonstrated that LySR activation boosts lysosomal activity.

Related technique Lysosome Detection, Lysosomal Acidic pH Detection

2. STING mediates lysosomal quality control and recovery through its proton channel function and TFEB activation in lysosomal storage disorders (Molecular Cell, 2025)

Summary: STING, long recognized as a cytosolic DNA sensor that initiates immune responses, is now shown to have a distinct role in maintaining lysosomal homeostasis. This study reveals that lysosomal dysfunction activates a STING–TFEB pathway, in which STING’s proton channel function triggers TFEB-driven expression of lysosomal and autophagy genes, thereby facilitating lysosomal repair and highlighting new therapeutic avenues for lysosomal storage disorders and neurodegenerative diseases.

Highlighted technique: To evaluate lysosomal damage and repair, researchers used LLOME to rupture lysosomal membranes. When lysosomes were damaged, cytosolic Galectin-3 bound to the exposed glycans and was visualized as fluorescent puncta with antibodies, allowing the appearance and disappearance of these puncta over time to reveal the repair process.

Related technique Lysosomal Function, Autophagic Flux Assay

All Related Techniques (click to open/close)

Target	Kit & Probes
Lysosomal function	Lysosomal Acidic pH Detection Kit -Green/Red and Green/Deep Red
First-time autophagy research	Autophagic Flux Assay Kit
Mitophagy detection	Mitophagy Detection Kit
Endocytosis Detection	ECGreen-Endocytosis Detection
Cellular senescence detection	SPiDER-βGal for live-cell imaging or flow cytometry / microplate reader / tissue samples Blue cellular senescence detection dye for fixed cells, SPiDER Blue
Mitochondrial superoxide detection	MitoBright ROS Deep Red - Mitochondrial Superoxide Detection
Total ROS detection	Highly sensitive DCFH-DA or 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 (click to open/close)
> Accurate Measurement for Lysosomal pH changes

With existing reagents, it was difficult to determine whether lysosomal mass or their function (pH) fluctuated because the discussion was based on changes in the fluorescence brightness of a single dye. This kit contains pHLys Green, which is highly specific to lysosomes and shows pH-dependent changes in fluorescence, and pH-resistant LysoPrime Deep Red. Using these two dyes, lysosomal pH and volume of the same sample can be measured for a detailed analysis of lysosomal function.
Existing lysosomal pH detection reagents have issues with dye localization, pH sensitivity, and retention. pHLys Green is a dye that solves these issues. The improved dye retention and localization enable detection of normal lysosomes, and the improved pH sensitivity enables detection of slight pH changes.
1. High sensitive pH detection Comparison of pH response of cells treated with low concentrations of lysosomal acidification inhibitor Bafilomycin A1	2. High specificity for lysosomes Comparison of specificity for lysosomes using lysosomal marker protein LAMP1-GFP expressing cells	3. High retention in lysosomes Comparison of intracellular retention

Product in Use: - Lysosomal Acidic pH Detection Kit-Green/Deep Red Related Product: - Lysosomal Acidic pH Detection Kit-Green/Red - LysoPrime Deep Red - High Specificity and pH Resistance - pHLys Red - Lysosomal Acidic pH Detection

Topics

What is Lysosome?
How to Analyze Lysosomal Function?
Lysosome Staining Reagents and Kits
Experimental Example: Effect of lysosomal acidification inhibitor on endocytic vesicle fusion with lysosome
Experimental Example: Effect of mitochondrial inhibitors on lysosomal function
Experimental Example: Measurement of intracellular iron changes and lysosomal pH changes

What is Lysosome?

Lysosomes are essential for maintaining cell homeostasis by degrading and recycling biomolecules, regulating organelle quality control, and facilitating intracellular signaling. Lysosomal function is closely linked to the Golgi apparatus, endoplasmic reticulum, mitochondria, and nucleus, coordinating cellular metabolism and stress responses. When lysosomal function is impaired, damaged proteins and organelles accumulate, metabolic processes are disrupted, and cell membrane integrity is compromised, leading to various diseases. For example, in neurodegenerative diseases, lysosomal dysfunction leads to the accumulation of toxic aggregates, resulting in neuronal damage and cognitive decline. Understanding lysosomal regulation and its interactions with other organelles is critical for developing therapies to slow disease progression and promote cellular longevity.

How to Analyze Lysosomal Function？

When conventional dyes are used to analyze lysosomal function, it is difficult to determine whether the lysosomal mass or their function (pH) has changed because the analysis is based only on the fluorescence intensity of a single dye.
Dojindo's kits contain two types of dyes: pHLys Red/Green, which shows a lysosomal pH-dependent change in fluorescence intensity, and LysoPrime Green/Deep Red, which is lysosomal pH-resistant. By combining these two dyes, the lysosomal function can be analyzed in detail by simultaneously analyzing lysosomal mass and pH.

Lysosome Staining Reagents and Kits

Explore Dojindo's wide range of lysosomal staining and pH detection dyes. Choose the following kit or reagent that aligns with your experimental requirements.

Product Name (Item Code)	Supported Devices			Indicator and Detection Color	Dyes and Fluorescence Properties	Approximate Number of Use
Product Name (Item Code)				Indicator and Detection Color	Dyes and Fluorescence Properties	Approximate Number of Use
Lysosomal Acidic pH Detection Kit-Green/Deep Red (L268)	✓	✓	✓	pH	pHLys Green Ex: 488 nm / Em: 490-550 nm	[for 1 set] 35 mm dish: 10 dishes μ-Slide 8 well: 10 plates 96-well Plate: 2 plates
Lysosomal Acidic pH Detection Kit-Green/Deep Red (L268)	✓	✓	✓	quantity	LysoPrime Deep Red Ex: 633 nm / Em: 640-700 nm
Lysosomal Acidic pH Detection Kit-Green/Red (L266)	✓	Need G/Y Laser G:532 nm Y:561 nm	✓	pH	pHLys Red Ex: 561 nm / Em: 560-650 nm
Lysosomal Acidic pH Detection Kit-Green/Red (L266)	✓		✓	quantity	LysoPrime Green Ex: 488 nm / Em: 500-600 nm
pHLys Red- Lysosomal Acidic pH Detection (L265)	✓		✓	pH	pHLys Red Ex: 561 nm / Em: 560-650 nm	[for 1 tube] 35 mm dish: 10 dishes μ-Slide 8 well: 10 plates 96-well Plate: 2 plates
LysoPrime Deep Red - High Specificity and pH Resistance (L264)	✓	✓	✓	quantity	LysoPrime Deep Red Ex: 633 nm / Em: 640-700 nm
LysoPrime Green- High Specificity and pH Resistance (L261)	✓	✓	✓	quantity	LysoPrime Green Ex: 488 nm / Em: 500-600 nm	[for 10 μl] 35 mm dish: 10 dishes μ-Slide 8 well: 10 plates 96-well Plate: 2 plates

Experimental Example: Effect of lysosomal acidification inhibitor on endocytic vesicle fusion with lysosome

Endocytic vesicles were labeled by ECGreen and the lysosomal mass and pH were detected separately with LysoPrime Deep Red and pHLys Red. Co-staining with ECGreen and Lysosomal dyes showed the inhibition of endocytic vesicle-fusion induced by Bafilmycin A1.

Experimental Example: Effect of mitochondrial inhibitors on lysosomal function

CCCP and Antimycin are recognized inducers of mitochondrial ROS, linked to the loss of mitochondrial membrane potential. Recent studies have shown that CCCP induces not only mitochondrial ROS but also lysosomal dysfunction. To observe mitochondrial ROS, HeLa cells were labeled with MitoBright ROS Deep Red for Mitochondrial Superoxide Detection, and the lysosomal mass and pH were independently detected with LysoPrime Green and pHLys Red. Co-staining with MitoBright ROS and Lysosomal dyes revealed that CCCP, unlike Antimycin, triggers concurrent lysosomal neutralization and mitochondrial ROS induction.

Reference: Benjamin S Padman, et. al., Autophagy (2013)

Products in Use
- LysoPrime Green
- pHLys Red
- Lysosomal Acidic pH Detection Kit
- MitoBright ROS Deep Red - Mitochondrial Superoxide Detection

Related Products
- Mitophagy Detection Kit and Mtphagy Dye

Experimental Example: Measurement of intracellular iron changes and lysosomal pH changes

In neurodegenerative diseases, the relationship between lysosomal function and iron has attracted attention, and it has been reported^* that lysosomal neutralization prevents the breakdown of iron stores (Transferrin or Ferritin), resulting in a decrease in intracellular iron.
Lysosomal pH changes and intracellular iron changes in the same sample were detected using SH-SY5Y cells supplemented with lysosomal acidification inhibitor (Bafilomycin A1) or iron chelator (Deferipron (DFP)). (Lysosomal pH: Lysosomal Acidic pH Detection kit - Green/Deep Red, Intracellular iron: FerroOrange [Code:F374])
The results showed that the addition of Bafilomycin A1 decreased the fluorescence of FerroOrange, confirming the decrease in intracellular iron. The fluorescence of LysoPrime DeepRed remained almost unchanged, while the fluorescence of pHLys Green decreased due to lysosomal neutralization. These results suggest that there is a relationship between changes in intracellular iron and lysosome function.

*Mol Cell., 2020, 77(3), 645-655.

＜Condition＞
pHLys Green (Green) : Ex=488 nm, Em=486-574 nm
FerroOrange (Red) : Ex=561 nm, Em=550-650 nm
LysoPrime Deep Red (Violet) : Ex=633 nm, Em=599-700 nm