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

Science Note

Lysosome Damage Responses in Disease Biology [Jun. 16, 2026] 

Previous Science Note

Lysosomes regulate degradation, membrane integrity, and immune-related processing. Lysosome damage responses are important for understanding how organelle injury influences disease-relevant cellular outcomes. Recent studies identified Parkinson's disease-associated VPS13C as an early factor recruited to damaged lysosomes, suggesting a membrane-protective response before severe rupture. In macrophages lacking TMEM175, lysosomal stress during tumor debris processing promoted inflammatory cytokine release and responses that help present tumor antigens to CD8⁺ T cells. These findings support lysosome damage responses as a relevant focus in disease research. 

1. The bridge-like lipid transport protein VPS13C/PARK23 mediates ER-lysosome contacts following lysosome damage
(Nature Cell Biology, 2025)

Summary: 
This study showed that the Parkinson’s disease associated protein VPS13C acts as an early response factor to lysosome damage. VPS13C was recruited to damaged lysosomes within minutes and mediated ER lysosome contacts, suggesting a membrane protective response before severe rupture marked by Gal3. Its distinct timing from LRRK2, a Parkinson’s disease associated kinase recruited later to stressed lysosomes, suggests VPS13C dysfunction may reduce early lysosome damage resilience. 

Highlighted technique: 
To assess lysosome homeostasis associated with VPS13C loss, the authors compared wild type and VPS13C-knockout A549 cells. Lysosome-associated LAMP1 signal was evaluated by LAMP1 immunofluorescence, while lysosome acidification was measured using a pH-sensitive lysosome probe, providing readouts of lysosome status before damage-challenge experiments.

By combining pH-dependent and pH-independent lysosome probes, lysosomal pH and mass can be assessed by fluorescence imaging without immunostaining or transfection.

2. Deficiency of lysosomal TMEM175 in myeloid macrophages exerts anti-tumor immunity via inflammasome and cross-presentation pathway
(Nature Communications, 2026)

Summary: 
TMEM175 deficiency in macrophages showed that lysosomal dysfunction can convert tumor cell debris processing into an anti-tumor immune signal. After debris uptake, increased lysosomal membrane permeabilization and cathepsin B leakage activated NLRP3 inflammasome signaling and IL-1β and IL-18 secretion. Delayed lysosomal antigen degradation likely supported enhanced cross-presentation, strengthening CD8⁺ T cell immunity. These findings suggest that lysosomal stress in macrophages supports tumor antigen presentation and CD8⁺ T cell-mediated immune surveillance.

Highlighted technique:
To examine whether lysosomal dysfunction promotes inflammasome activation after tumor debris uptake, the authors exposed macrophages to B16-F10 tumor cell debris. They assessed lysosomal membrane damage by electron microscopy and cathepsin B immunofluorescence, measured Ca²⁺ changes and intracellular ROS with fluorescent probes, and evaluated NLRP3 activation by cytokine release and protein interaction assays.

Evaluating Ca²⁺ dynamics and intracellular ROS helps clarify lysosome-associated inflammasome activation. As a complementary approach, detecting intracellular and lysosomal lipid radicals may provide additional information on oxidative membrane stress associated with lysosomal dysfunction. 

 
All Related Techniques (click to open/close)
Target Kit & Probes
Lysosomal Function Analysis Kit Lysosomal Acidic pH Detection Kit - Green/Red and Green/Deep Red
High Specific Lysosommal Detection LysoPrime Green / Deep Red
Lysosomal Acidic pH Detection pHLys Red
Intracellular Calcium detection Calcium Kit II - Fluo 4 and Calcium Kit II - Fura 2
Total ROS detection Highly sensitive DCFH-DA or Photo-oxidation Resistant DCFH-DA
Intracellular lipid radical detection Lipid Radical Probe -NBD-Pen-
Lysosomal lipid radical detection Lysosomal Lipid Radical Probe -Lyso-NBD-Pen-
Lipid Peroxidation Assay Lipid Peroxidation Probe -BDP 581/591 C11-
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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