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

Science Note

[Jan. 3, 2024]                                                                                                                                                                                                                            Previous Science Note
Lysosomes in the Regulation of Metabolism and Organelle

Lysosomal dysfunction is increasingly recognized as a critical factor in the development and progression of several neurological diseases. In neurodegenerative diseases such as Parkinson's and Alzheimer's, impaired lysosomal function leads to the accumulation of misfolded proteins and neuronal toxicity, contributing to cell death and disease progression. Although rare, lysosomal storage diseases often have significant neurological manifestations due to the accumulation of undigested substances in neurons, impairing their function and survival. Therefore, understanding and targeting lysosomal pathways is emerging as a promising therapeutic approach to treat and potentially prevent a range of neurological disorders. 

Lysosomes mediate the mitochondrial UPR via mTORC1-dependent ATF4 phosphorylation
Click here for the original article: Terytty Yang Li, et. al., Cell Discovery, 2023.

ARL8B mediates lipid droplet contact and delivery to lysosomes for lipid remobilization
Click here for the original article: Dilip Menon, et. al., Cell Reports, 2023.

Nutrient-regulated control of lysosome function by signaling lipid conversion
Click here for the original article: Michael Ebner, et. al., Cell, 2023.

Point of Interest
- Mitochondrial stress activates mTORC1 through v-ATPase-mediated lysosomal stimulation, leading to phosphorylation of ATF4.

- This phosphorylation of ATF4 triggers the mitochondrial unfolded protein response (UPRmt).

- Interfering with the ability of mTORC1 to phosphorylate ATF4 inhibits the UPRmt.

- The phosphorylation of ATF4 prevents ROS-induced cell death under mitochondrial stress.

Point of Interest
- ARL8B enhances the lysosomal lipolysis of triacylglycerol stored in lipid droplets (LDs).

- ARL8B in its GDP-bound state associates with LDs, whereas ARL8B-GTP is predominantly associated with lysosomes.

- ARL8B facilitates the contact between LDs and lysosomes, with both its GDP- and GTP-bound states forming a complex.

- The ARL8B-mediated lysosomal lipolysis of LDs represents a pathway for the turnover of neutral lipids in macrophages.

Point of Interest
- Starvation enhances lysosomal catabolism through the localized synthesis of PI(4)P on the lysosomal membrane.

- Cells possess diverse lysosome populations, distinguished by the presence of either PI(3)P or PI(4)P.

- A lipid switch, regulated by nutrient availability, facilitates the interconversion between these distinct lysosome populations.

- mTORC1 signaling and the synthesis of lysosomal PI(4)P are mutually inhibitory, each suppressing the other's activity.

Related Techniques
           Lysosomal function Lysosomal Acidic pH Detection Kit-Green/Red and Green/Deep Red NEW
           Autophagy detection DAPGreen / DAPRed (Autophagosome detection), DALGreen (Autolysosome detection) HOT
           Endocytosis detection ECGreen-Endocytosis Detection
           Phagocytosis detection AcidSensor Labeling Kit – Endocytic Internalization Assay and -Cellstain- Calcein-AM solution
           Mitophagy Detection Mitophagy Detection Kit and Mtphagy Dye
           Mitochondrial superoxide detection mtSOX Deep Red
           Oxygen consumption rate assay Extracellular OCR Plate Assay Kit
           Antibody/Protein labeling - fast and high recovery Fluorescein, Biotin, and Peroxidase Labeling Kit - NH2
The products listed above are currently on special promotional offer.
Related Applications

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 mtSOX Deep Red for Mitochondrial Superoxide Detection, and the lysosomal mass and pH were independently detected with LysoPrime Green and pHLys Red. Co-staining with mtSOX 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
   - mtSOX Deep Red - Mitochondrial Superoxide Detection

Related Products
   - Mitophagy Detection Kit and Mtphagy Dye


Induction of Ferroptosis by Erastin

Erastin is a known inducer of ferroptosis. By inhibiting the cystine transporter (xCT), erastin inhibits the uptake of cystine. Cystine is the raw material for GSH. Therefore, Erastin ultimately decreases the amount of GSH. Decreased GSH then results in lipid peroxide accumulation and induction of ferroptosis.
The following experimental examples show changes in each aforementioned index as a consequence of erastin stimulation. Measurements are made using Dojindo reagents.

Using erastin-treated A549 cells, we measured intracellular Fe2+, ROS, lipid peroxide, glutathione, glutamate release into the extracellular space, and cystine uptake. As a result, inhibition of xCT by elastin was observed and also the release of glutamate and uptake of cystine were decreased. Furthermore, elastin treatment decreased intracellular glutathione while it increased intracellular Fe2+ , ROS, and lipid peroxides.

①Cystine Uptake     

Cystine Uptake Assay Kit

Released Glutamate

Glutamate Assay Kit

Intracellular Glutathione

GSSG/GSH Quantification Kit

④Intracellular Fe2+

FerroOrange

⑤Intracellular ROS

Highly Sensitive DCFH-DA

⑥Intracellular Lipid

Liperfluo

 

 


 

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 mtSOX Deep Red - Mitochondrial Superoxide Detection, and the lysosomal mass and pH were detected separately with LysoPrime Green and pHLys Red. Co-staining with mtSOX 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

Product Classification

Product Classification