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

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.   Master the Basics with a Overview Map!
      
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Science Note

[Apr. 29, 2025]                                                                                                                                                                                                                   Previous Science Note

The Role of Lysosomal Dysfunction in Cell Death  

Lysosomes and cell death pathways are fundamental to the regulation of cellular homeostasis, yet their dysregulation can contribute to pathological conditions. Recent studies highlight how impaired lysosomal function can trigger various forms of cell death. This Science Note introduces emerging insights into lysosomal dynamics and the mechanisms of cell death.

Lysosomal lipid peroxidation contributes to ferroptosis induction via lysosomal membrane permeabilization (Nature Communications, 2025)

Summary: The accumulation of lipid peroxidation within lysosomes increases lysosomal membrane permeability, leading to the release of iron into the cytosol and subsequently triggering ferroptosis. Increasing lysosomal membrane permeability may be a promising strategy to overcome ferroptosis resistance in certain cell types.

Highlighted technique: In this study, lipid peroxide accumulation within lysosomes was visualised following induction of ferroptosis. In addition, increased lysosomal pH, cytosolic diffusion of internalised FITC-dextran and iron redistribution from lysosomes to the cytosol were observed, demonstrating that lysosomal membrane permeabilization contributes to ferroptosis.

 Related technique   Accurate lysosomal detection / Fe2+ live-imaging (used in this article) / Lipid peroxide detection  (used in this article)

Proteostasis and lysosomal repair deficits in transdifferentiated neurons of Alzheimer’s disease (Nature Cell Biology, 2025)

Summary: This study develops tNeurons, an aged human neuron model, and shows that lysosomal dysfunction in these cells drives amyloid β accumulation, inflammation and cell death, highlighting tNeurons as a powerful platform to study early pathogenic events and lysosomes as a therapeutic target in Alzheimer's disease.

Highlighted technique: tNeurons are human neurons directly converted from aged dermal fibroblasts without transitioning through a pluripotent state, preserving age-associated dysfunctions such as lysosomal and mitochondrial impairments and disrupted proteostasis. They provide a valuable model for studying the progression of age-related diseases, including Alzheimer’s disease.

 Related technique   Accurate lysosomal detection / Senescence assay / Apoptosis plate assay

Faulty autolysosome acidification in Alzheimer’s disease mouse models induces autophagic build-up of Aβ in neurons, yielding senile plaques (Nature Neuroscience, 2022)

Summary: In Alzheimer's disease models, impaired lysosomal acidification causes Aβ and amyloid precursor protein-beta C-terminal fragment (APP-βCTF) accumulation in autolysosomes. This dysfunction leads to the formation of PANTHOS, a flower-like aggregation of autophagic vacuoles, along with lysosomal membrane permeabilization and cell death, promoting senile plaque development.

Highlighted technique: In this study, neuronal autolysosomal acidification and dysfunction were assessed using a neuron-specific mRFP-eGFP-LC3 probe, where the pH-sensitive eGFP signal is quenched while the pH-stable mRFP signal persists in acidic compartments, enabling detection of acidification defects by multiplex confocal and correlative light-electron microscopy.

 Related technique   Accurate lysosomal detection / Autophagic Flux Assay Kit
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
Fe2+ detection FerroOrange(intracellular), Mito-FerroGreen(mitochondria)
Lipid peroxidation detection Liperfluo(intracellular), MitoPeDPP(mitochondria)
Apoptosis detection in multiple samples  Annexin V Apoptosis Plate Assay Kit
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 membrane potential detection  JC-1MitoMPDetection Kit, MT-1MitoMPDetection 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
 

What is Lysosome?

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
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
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
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

 

 

 

 

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