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

Science Note

Lysosomal Control of Neuron Glia Crosstalk [Mar. 31, 2026] 

Previous Science Note

Lysosome dependent degradation in neurons and surrounding glial cells is essential for maintaining neural homeostasis under stress. Clarifying how these pathways process protein aggregates and lipid burden is important for understanding mechanisms of neuronal protection and intercellular responses in the nervous system. Recent studies showed that exposure of neurons to aggregated α-synuclein impairs lysosomal and autophagic function, thereby promoting tunneling nanotube mediated transfer of aggregates to microglia. Another study showed that the endolysosomal protein TTYH1 supports astrocytic autophagic flux and lipid droplet degradation under inflammatory stress, contributing to metabolic adaptation to neuron derived lipid signals. Together, these findings identify glial lysosome related pathways as one component of neuron protective responses.

1. Impaired α-Synuclein aggregate clearance in neuronal cells drive their spread to microglia through tunneling nanotubes (Nature Communications, 2026)

Summary: Aggregated α-synuclein is a key feature of neurodegenerative diseases and can transfer from neurons to microglia through tunneling nanotubes. This study demonstrates that impaired autophagy in neurons reduces their ability to degrade α-synuclein aggregates, thereby promoting their TNT-mediated transfer to microglia. It further reveals that microglia possess higher lysosomal turnover and efficiently clear the transferred aggregates. Altogether, the findings identify dysfunctional neuronal autophagy as a central driver of the directional spread of α-synuclein aggregates toward microglia.

Highlighted technique: To determine whether differences in degradative capacity between neurons and microglia drive the directional transfer of α-synuclein aggregates, the authors exposed each cell type to α-synuclein aggregates and assessed their intracellular handling using AlexaFluor647-conjugated Dextran for lysosome tracking and DQ-BSA for lysosomal degradative activity measurements. In addition, they evaluated autophagic flux using p62 puncta, which can reflect either increased autophagosome formation or impaired cargo degradation, in the presence or absence of bafilomycin A1.

2. Endolysosomal processing of neuron-derived signaling lipids regulates autophagy and lipid droplet degradation in astrocytes (Nature Communications,, 2025)

Summary: Neuronal lipid handling depends in part on surrounding astrocytes, and this study shows that the endolysosomal protein TTYH1 is a key factor supporting autophagic flux and lipid droplet degradation in astrocytes. Under inflammatory stress, neuron-derived ceramide 1-phosphate signaling places a burden on these astrocytic endolysosome-dependent degradative pathways, which is alleviated by TTYH1-dependent endolysosomal clearance.

Highlighted technique: The authors used primary astrocytes expressing the tandem reporter GFP-LC3-RFP-LC3ΔG to monitor autophagic flux under starvation conditions in the presence or absence of TTYH1. In this reporter, GFP-LC3 is degraded by autophagy whereas RFP-LC3ΔG remains in the cytosol as an internal control, allowing the GFP/RFP signal ratio to be used as a readout of autophagic flux.

Solutions for Lysosome Experiments
To facilitate lysosome research, a lysosome-tracking dye with fluorescence that is more pH-stable than widely used commercial reagents, along with a more sensitive lysosomal pH probe probe than conventional pH dyes, provides reliable tools. See the Performance Comparison
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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
 

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!
      
(Click to open)

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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