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

Science Note

[Jul. 30, 2024]                                                                                                                                                                                                                            Previous Science Note
Collapse of Lysosomal Function is a Trigger of Cancer, Neurodegeneration and Senescence

Lysosomal dysfunction plays a critical role in several diseases, including cancer, where it can promote tumor growth and resistance to therapy by altering cellular metabolism and autophagy. In neurodegenerative diseases such as Alzheimer's and Parkinson's, impaired lysosomal activity leads to the accumulation of toxic proteins, contributing to neuronal death and disease progression. Lysosomal dysfunction is also associated with cellular senescence, where it impairs the degradation of damaged cellular components, leading to the accumulation of senescent cells and promoting age-related diseases. Therapeutic strategies targeting lysosomal function are being explored to alleviate these conditions and improve overall cellular health. 

Lysosomal cystine governs ferroptosis sensitivity in cancer via cysteine stress response
Click here for the original article: Robert Swanda, et. al., Molecular Cell, 2023.

Glucosylceramide accumulation in microglia triggers STING-dependent neuroinflammation and neurodegeneration in mice
Click here for the original article: Rui Wang, et. al., Science Signaling, 2024.

HKDC1, a target of TFEB, is essential to maintain both mitochondrial and lysosomal homeostasis, preventing cellular senescence
Click here for the original article: Mengying Cui, et. al., PNAS, 2023.

Point of Interest
- Lysosomal cystine depletion, but not cytosolic cysteine, induces ATF4, a survival factor for many cancer cells, and suppresses ferroptosis.

- The AhR, which is a lysosomal cystine sensing receptor, influences ATF4 response and ferroptosis sensitivity.

- Conversion of cytosolic cysteine to lysosomal cystine by a synthetic mRNA reagent enhances ferroptosis in cancer cells and suppresses tumor growth.

Point of Interest
- Genetic mutations of the lysosomal enzyme glucocerebrosidase (GCase) lead to the accumulation of glucosylceramides (GCs) in cells.

- GC accumulation in microglia triggers stimulator of interferon genes (STING)-dependent inflammation and neuronal loss by causing mtDNA leakage, and this process has been implicated in Gaucher disease, Parkinson's disease, and Lewy bodies.

- Rapamycin, a compound that promotes lysosomal activity, improves mitochondrial function and reduces STING signaling, potentially suppressing GCase-deficiency-induced neuroinflammation.

Point of Interest
- Mitochondrial and lysosomal functions are linked, with transcription factor EB (TFEB) and hexokinase domain containing 1 (HKDC1) playing critical roles in their regulation and maintenance.

- HKDC1, regulated by TFEB, is essential for mitophagy and lysosomal repair, independent of its glycolysis function.

- Loss of HKDC1 accelerates cellular senescence by causing mitochondrial hyperfusion and lysosomal damage
Related Techniques
           Lysosomal function Lysosomal Acidic pH Detection Kit-Green/Red and Green/Deep Red
           Cystine Uptake detection Cystine Uptake Assay Kit
           Ferrous ion (Fe2+) detection FerroOrange(intracellular), Mito-FerroGreen(mitochondrial)
           Lipid peroxidation detection Liperfluo(intracellular), MitoPeDPP(mitochondrial)
           Cellular senescence detection SPiDER-βGal for live-cell imaging or flow cytometry / microplate reader / tissue samples.
           Mitophagy or autophagy detection Mitophagy Detection Kit, Autophagic Flux Assay Kit
           Mitochondrial membrane potential detection JC-1 MitoMP Detection Kit,  MT-1 MitoMP Detection Kit
           Glycolysis/Oxidative phosphorylation Assay Glycolysis/OXPHOS Assay Kit
 
Related Applications

Accurate Measurement for Lysosomal pH changes

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.

Product in Use: Lysosomal Acidic pH Detection Kit-Green/Deep Red

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

 

The simultaneous detection of lysosomal function with Mitochondrial ROS and intracellular Fe2+

Lysosomal Function and Iron Homeostasis

 

 

 

Recent reports suggest that lysosomal neutralization can result in iron depletion, consequently leading to the disruption of cell viability. To verify this, HeLa cells were labeled with FerroOrange for Fe2+ detection, and the lysosomal mass and pH were separately detected with LysoPrime DeepRed and pHLys Green (a product currently under development). Co-staining with FerroOrange and Lysosomal dyes demonstrated that Bafilomycin A1 (Baf. A1), an inhibitor of lysosomal acidification, causes iron depletion consistent with the findings reported in the article. Interestingly, the iron chelator, Deferiprone (DFP), did not impact lysosomal pH, suggesting that lysosomal function plays a key role in managing iron homeostasis.
Reference: Ross A Weber, et. al., Mol Cell (2020)

Products in Use
   - FerroOrange
   - pHLys Green*
   - LysoPrime Deep Red

*pHLys Green is included as a component of the "Lysosomal Acidic pH Detection Kit-Green/Deep Red".

Lysosomal Function and Mitochondrial ROS

 

 

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

 

 

 

Why is Lysosomal Function Important?

The 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.


Lysosomal pH Detection Dye: pHLys Red
pHLys Red has a high sensitivity to lysosomal pH change with more precise localization, which allows slight pH changes to be detected in physiological conditions like the early phase of the disease.

 

 

Lysosomal Mass Detection Dye: LysoPrime Green, Deep Red
Dojindo’s LysoPrime Green / Deep Red more precisely detects lysosomal mass due to their unique specificity for lysosomes, and retention independent of lysosomal pH. In addition, the retention time of the dyes in lysosomes has been extended, allowing data to be acquired over time.

 

Recommended for First Time Users
Lysosomal Acidic pH Detection Kit
Two types of dyes and lysosomal pH acidification inhibitors are available as a set. Positive control is included for immediate testing in your experimental system.


- Lysosomal mass detecting dye: LysoPrime Green
- Lysosomal pH detection dye: pHLys Red
- Lysosomal pH acidification inhibitor: Bafilomycin A1

Application data

 

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.

 

We tried the simultaneous detection of lysosomal and mitochondrial dysfunction in Hela cells treated with CCCP or Antimycin (AN). CCCP and AN are well-known inducers of mitochondrial ROS regarding loss of mitochondrial membrane potential. Recent research showed the result that CCCP induces not only mitochondrial ROS but also lysosomal neutralization. To detect mitochondrial ROS, HeLa cells were labeled by MitoBright ROS Deep Red - Mitochondrial Superoxide Detection, and the lysosomal mass and pH were detected separately with LysoPrime Green and pHLys Red. Co-staining with MitoBright ROS and Lysosomal dyes demonstrated that CCCP causes lysosomal neutralization and mitochondrial ROS induction at the same time.

 

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

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