Mitochondria Research

Science Note

Iron-Related Autophagy and Mitochondrial Homeostasis 

Scientists have unveiled that the selective autophagy adaptor NCOA4, which targets ferritin (a process known as ferritinophagy), is upregulated in pancreatic cancer. This upregulation ensures the availability of iron, thus encouraging tumor progression. Fascinatingly, ferritinophagy facilitates the synthesis of iron–sulfur cluster proteins, which help maintain mitochondrial homeostasis. Discover how the authors employed separate iron-detecting probes: FerroOrange for the cytosol and Mito-FerroGreen for the mitochondria (refer to Figure 1I and Supplemental Figure S4J).

NCOA4-Mediated Ferritinophagy Is a Pancreatic Cancer Dependency via Maintenance of Iron Bioavailability for Iron–Sulfur Cluster Proteins    Naiara Santana-Coodina, et. al., Cancer Discovery (2022)

Point of Interest
- Ferritinophagy, the process by which iron is released from storage for use in cells, is found to be upregulated in pancreatic ductal adenocarcinoma (PDAC), promoting tumor growth.
- It is shown to support the synthesis of iron–sulfur cluster proteins, thereby maintaining mitochondrial stability.
- Blocking the ferritinophagy adapter NCOA4 delays tumor growth and extends survival, although compensatory iron-gathering pathways may develop.
Related Techniques
Intracellular ferrous ion (Fe2+) detection FerroOrange
Mitochondria ferrous ion (Fe2+) detection Mito-FerroGreen
Mitophagy Detection Mitophagy Detection Kit
​​Lysosomal function assay Lysosomal pH and mass detection Kit
Mitophagy Detection Mitophagy Detection Kit and Mtphagy Dye
Autophagy detection DAPGreen / DAPRed (Autophagosome detection), DALGreen (Autolysosome detection)​
Oxygen consumption rate assay Extracellular OCR Plate Assay Kit
Related Applications

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

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

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 - Mitochondrial Superoxide Detection

Mitochondria Research

Mitochondria, which have important functions in cells, are not only a place for ATP production by oxidative phosphorylation, but their activity and dysfunction are closely related to cancer, senescence, and neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Therefore, understanding the state of mitochondria is particularly important.

 

Mitophagy

Target Mitophagy
Reagent Mitophagy Detection Kit Keima-Red
Priciple Mtphagy Dye (included in Mitophagy Detection Kit) is a pH-sensitive fluorescent probe that accumulates in mitochondria and emits red fluorescence due to acidic conditions in a lysosome. A pH-sensitive ratiometric fluorescent protein. The excitation spectrum changes according to pH. This protein shows high fluorescence ratio (Ex. 550 nm/440 nm) values in a lysosome.
Accumulating in mitochondria Yes
（Covalent bond）		 -
（Expression protein）
Live-cell staining
（Fixation after live-cell staining）		 ✓
(-)		 ✓
(-)
Fixed cell staining - -
Ex/Em (nm) 530/700  550 & 440/620
Staining time*1 30 min～ *2
Product code MD01 -

*1  Suitable staining time depends on sample type and reagent concentration.
*2  It is necessary to construct a keima expression strain.

Oxidative Stress

  Target Lipophilic Peroxide ROS Singlet oxygen Superoxide
Reagent MitoPeDPP ROS Assay Kit Si-DMA MitoBright ROS
Deep Red		 MitoSOX Red
Principle A cell-permeant fluorescent probe that accumulates in mitochondria and specifically reacts with lipophilic peroxides in mitochondria to emit fluorescence. A cell-permeant fluorescent probe that  reacts with ROS  generated in mitochondria to emit fluorescence. A cell-permeant fluorescent probe that accumulates in mitochondria and specifically reacts with singlet oxigen generated in mitochondria to emit fluorescence. A cell-permeant fluorescent probe that accumulates in mitochondria and reacts with superoxide generated in mitochondria to emit fluorescence. A cell-permeant fluorescent probe that accumulates in mitochondria and reacts with superoxide generated in mitochondria to emit fluorescence.
Accumulating in mitochondria Yes
（Electrostatic interaction）		 - Yes
（Electrostatic interaction）		 Yes
（Electrostatic interaction）		 Yes
（Electrostatic interaction）
Live-cell staining
（Fixed Cell staining）		 ✓
(-)		 ✓
(-)		 ✓
(-)		 ✓
(-)		 ✓
(-)
Fixed cell staining - - - - -
Ex/Em(nm) 452/470 505/525 644/670 540/670 510/580
Common filter set FITC FITC Cy5 TexasRed
Cy5		 TRITC
Staining time*1 30 min～ 15 min～ 45 min～ 10 min~ 10 min～
Product Code M466 R252 MT05 MT16 -

*1  Suitable staining time depends on sample type and reagent concentration.

Mitochondrial Membrane Potential

Target Mitochondrial Membrane Potential
Reagent MT-1 JC-1
Principle Cell-permeant fluorescent dyes accumulate in intact mitochondria due to the membrane potential. MT-1 is extremely photostable and more sensitive than JC-1 and can provide equivalent detection sensitivity to TMRE. A cell-permeant ratiometric fluorescent dye that accumulates in intact mitochondria due to the membrane potential. JC-1 forms aggregate (in healthy mitochondria) with red fluorescence.
As membrane potential decreases, JC-1 becomes monomers, which shows in green fluorescence.
Live-cell staining
（PFA fixation after staing）		 ✓
(✓)		 ✓
(-)
Fixed cell staining - -
Ex/Em(nm) 530-560/570-640 Monomer: 514/529
J-aggregation: 585/590
Common filter set TRITC
TexasRed		 Monomer: FITC
J-aggregation: TRITC; TexasRed
Staining time*1 30  min 10～60  min
Product Code MT13 MT09

*1  Suitable staining time depends on sample type and reagent concentration.

Mitochondria Staining

Target Localization of mitochondria
Reagent MitoBright LT series MitoBright IM Red MitoTracker series Rhodamine 123
Principle

Cell-permeant fluorescent dyes accumulate in intact mitochondria due to the membrane potential.

Cell-permeant fluorescent dyes accumulate in intact mitochondria due to the membrane potential.

Cell-permeant fluorescent dyes accumulate in intact mitochondria due to the membrane potential.

Cell-permeant fluorescent dyes accumulate in intact mitochondria due to the membrane potential.
Live-cell staining
（PFA fixation after staining）		 ✓
(✓)		 ✓
(✓)		 ✓
(✓)		 ✓
(-)
Fixed cell staining - - - -
Combination with immunostaining - ✓ Conditionally -
Long-term retention ✓ × × ×
Ex/Em (nm) Green: 493/508
Red: 547/563
Deep Red:643/663		 549/566 490/516 ～ 644/665 507/529
Common filter set Green: FITC

TRITC

-

FITC
Red: TRITC
DEEP Red: Cy5
Staining time*1 10 min～ 30 min～ 15～45 min 15 min～
Product Code MT10　　MT11　　MT12 MT15 - R233

*1  Suitable staining time depends on the sample type and reagent concentration.

Metal Ion Detection

Target Calcium ion (Ca2+) Iron ion(Fe2+)
Reagent Rhod 2-AM Mito-FerroGreen
Principle A cell-permeant fluorescent probe that accumulates in mitochondria and specifically reacts with calcium ion in mitochondria to emit red fluorescence. A cell-permeant fluorescent probe that accumulates in mitochondria and specifically reacts with ferrous ion in mitochondria to emit green fluorescence.
Accumulating in mitochondria ✓
(Electrostatic interaction)		 ✓
(Electrostatic interaction)
Live-cell staining
（PFA fixation after staing）		 ✓
(-)		 ✓
(-)
Fixed cell staining - -
Ex/Em(nm) 553/576 505/535
Common filter set TRITC FITC
Staining time*1 30～60  min 30 min～
Product Code R002 M489

*1  Suitable staining time depends on the sample type and reagent concentration.

Product Classification

Product Classification

Search word