Ferroptosis: Mechanisms in Disease and Kit Selection

Why is ferroptosis research important?

Detection of ferroptosis is essential to elucidate its impact on neurodegenerative diseases and cancer, as it plays a role in neuronal loss in diseases such as Alzheimer's and Parkinson's, while also representing a potential therapeutic target in malignancies. Reliable detection of ferroptosis supports the development of neuroprotective strategies to slow disease progression and improves cancer treatment approaches by promoting ferroptotic cell death in therapy-resistant tumors.   Master the Basics with a Overview Map!
      
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Science Note

Ferroptotic Cells Trigger Nearby Cell Death [Apr. 15, 2025] 

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Ferroptotic cells release signals that spread beyond themselves, inducing lipid peroxidation and death in neighbouring cells. This Science Note introduces recent advances in our understanding of how signals from ferroptotic cells spread and the molecular mechanisms underlying this process.

Ferroptosis spreads to neighboring cells via plasma membrane contacts 
(Nature Communications, 2025)
Ferroptosis spreads to neighbouring cells through the transfer of lipid peroxidation across closely adjacent plasma membranes. This spread of lipid peroxidation is dependent on the presence of extracellular iron.

Highlighted technique: In this study, the authors developed a novel model for ferroptosis induction by creating cells in which the lipid peroxide-detoxifying enzyme GPX4 can be selectively depleted by optogenetic activation. This system allowed the induction of ferroptosis in specific cells, allowing the observation of lipid peroxidation and the spread of ferroptosis to neighbouring cells.

 Related technique  Lipid Peroxidation Detection, Intracellular Iron Detection

Emergence of large-scale cell death through ferroptotic trigger waves
(Nature, 2024)
This study shows that ferroptosis propagates as a trigger wave at a constant speed (~5.5 μm/min) over distances ≥5 mm, driving large-scale cell death seen in development and disease. ROS released during ferroptosis spread to neighboring cells and induce further death, sustained by positive feedback loops that amplify ROS production.

Highlighted technique: The propagation of ferroptosis was analysed by dynamic observation using a combination of intracellular ROS staining and dead cell staining. Real-time time-lapse fluorescence microscopy revealed that ROS generation from ferroptotic cells preceded the wave-like spread of cell death.

 Related technique  Highly Sensitive ROS Detection Dye, Long-Term ROS Detection Dye

Microglial ferroptotic stress causes non-cell autonomous neuronal death
(Molecular Neurodegeneration, 2024)
Ferroptotic stress in microglia initiates an inflammatory cascade that promotes the transformation of astrocytes into a neurotoxic state, ultimately leading to neuronal death. This pathway highlights a mechanism by which localised oxidative stress in glial cells can contribute to wider neurodegeneration.

Highlighted technique: In this study, ferroptosis inducers were applied to microglia, astrocytes or mixed glial cultures and the resulting conditioned media were added to neurons to assess lipid peroxidation and cell viability. Neuronal death was only observed with media from ferroptosis-treated mixed glial cultures, indicating that neurotoxicity requires interaction between multiple glial cell types.

 Related technique  Lipid Peroxidation Assay, Cell Viability Assay
Related Techniques (click to open/close)
Target Kit & Probes
Ferroptosis Indicator: ferrous ion (Fe2+) FerroOrange(intracellular), Mito-FerroGreen(mitochondria)
Ferroptosis Indicator: lipid peroxidation Liperfluo(intracellular), MitoPeDPP(mitochondria)
Lipid Peroxidation Assay Lipid Peroxidation Probe -BDP 581/591 C11-
Total ROS detection Highly sensitive DCFH-DA or Photo-oxidation Resistant DCFH-DA
Glutathione Quantification GSSG/GSH Quantification Kit
Cystine Uptake detection Cystine Uptake Assay Kit
MDA detection MDA Assay Kit
Mitochondrial superoxide detection MitoBright ROS Deep Red - Mitochondrial Superoxide Detection
Apoptosis detection in multiple samples Annexin V Apoptosis Plate Assay Kit
Cell proliferation/ cytotoxicity assay Cell Counting Kit-8 and Cytotoxicity LDH Assay Kit-WST
Application Note (click to open/close)
  > Erastin-Induced Ferroptosis: Evaluating Intracellular Uptake and Redox Balance

We investigated the transition of cellular metabolisms in A549 cells treated with erastin, a known ferroptosis inducer. Our results revealed the following.

Results
- The inhibition of cystine uptake by erastin led to a depletion of cysteine, which in turn increased the compensatory uptake of other amino acids.
- Glucose uptake, which typically promotes ferroptosis*, was found to decrease upon erastin treatment, suggesting a potential cellular self-defense mechanism.
- The depletion of cysteine resulted in a decrease in glutathione levels and an increase in Fe2+, ROS, and lipid peroxides, all of which are recognized markers of ferroptosis.

  Cell Line: A549
  Incubation Conditions: 100 μmol/l Erastin/MEM, 37℃, 3h
  *Reference: Xinxin Song, et al., Cell Reports, (2021)

Products in Use
① Amino Acid Uptake: Amino Acid Uptake Assay Kit
② Glucose Uptake: Glucose Uptake Assay Kit-Green
③ Cystine Uptake: Cystine Uptake Assay Kit
④ Intracellular glutathione: GSSG/GSH Quantification Kit
⑤ Intracellular labile Fe: FerroOrange
⑥ Intracellular total ROS: ROS Assay Kit -Highly Sensitive DCFH-DA-
⑦ Lipid Peroxides: Liperfluo
   
 

What is Ferroptosis?

“Ferroptosis” was coined by Stockwell et al. at Columbia University in 2012 and described as a form of iron-dependent cell death. * It was reported to be a form of programmed cell death by the Nomenclature Committee on Cell Death (NCCD) in 2018.
Ferroptosis is a form of programmed cell death caused by iron ion-dependent accumulation of lipid peroxides. Ferroptosis has been shown to follow a different cell death pathway from apoptosis and thus is attracting attention as a new target for cancer therapy. It has also been found to be associated with various diseases, such as neurodegenerative diseases, cerebral apoplexy, and hepatitis (MASH).

*S. J. Dixon, B. R. Stockwell, et al., Ferroptosis: an iron-dependent form of nonapoptotic cell death., Cell, 2012, 149(5), 1060.
 

How Does Ferroptosis Cause Cell Death?

Ferroptosis is characterized by the accumulation of lipid peroxides. Lipid peroxides are formed from oxidation of polyunsaturated fatty acids (PUFA) in membrane phospholipids, with iron suggested to be involved. Intracellular glutathione peroxidase 4 (GPX4) uses reduced glutathione (GSH), an antioxidant, to reduce lipid peroxides generated by reactive oxygen species (ROS).*
However, when lipid peroxides accumulate due to GPX4 disruption or GSH depletion, ferroptosis is triggered.

*Stockwell et al, a leading researcher in the field of ferroptosis, summarized inhibitors, inducers, and detection indicators of ferroptosis in the following review, in which Dojindo’s Liperfluo is introduced for detection of lipid peroxides.

B. R. Stockwell, et al., "Ferroptosis: A Regulated Cell Death Nexus Linking Metabolism, Redox Biology, and Disease.", Cell, 2017, 171, 273.

 

Research on Related Diseases

Metabolic dysfunction-associated steatohepatitis (MASH)

Suppression of hepatitis via ferroptosis

In a study involving the livers of MASH model mice, it was confirmed that necrosis precedes apoptosis in the development of fatty liver. Further experiments showed that ferroptosis is involved within necrosis as a trigger for steatohepatitis and that inhibition of ferroptosis almost completely suppressed the onset of hepatitis.

Minoru Tanaka, et al., "Hepatic ferroptosis plays an important role as the trigger for initiating inflammation in nonalcoholic steatohepatitis", Cell Death & Disease, 2019, 10, 449.

Related article: changes in intracellular markers associated with MASH

The article summarizes reports on changes in each indicator of metabolic states and cellular senescence using the NASH model.

(Click on the “MASH” tab in the link)

Experimental example: measurement of intracellular metabolism in MASH model tissue

Measurement of ATP, a-KG, and NAD levels in liver tissue of high-fat diet-treated type 1 diabetic model mice. (Please refer to each product’s website for more information, “Experimental Example: Change in Metabolism in Liver Tissue of MASH-Induced Mouse”)
Neurodegenerative disease

Confirmation of the link between lysosomal disorders and ferroptosis

In experiments using human neurons, it is reported that knockdown of the lysosomal protein prosaposin induces formation of lipofuscin, a hallmark of aging. This process involves the iron-catalyzed generation of reactive oxygen species, leading to induction of ferroptosis.

Martin Kampmann, et al., "Genome-wide CRISPRi/a screens in human neurons link lysosomal failure to ferroptosis", Nature Neuroscience, 2021, 24, 1020
Cancer

Regulation of cancer immunity via ferroptosis

CD8+ T cells activated by immunotherapy were found to confer an anti-tumor effect by promoting lipid peroxidation and inducing ferroptosis. The mechanism of immunotherapy-induced inhibition of cystine uptake and promotion of lipid peroxidation in tumor cells is discussed.

Weiping Zou, et al, "CD8+ T cells regulate tumour ferroptosis during cancer immunotherapy", Nature, 2019, 569, 270

Ferroptosis-Related Reagent Selection Guide

Lipid Peroxide and Iron (Fe2+) Detection Reagents

Name Liperfluo MitoPeDPP

 Lipid Peroxidation Probe
-BDP 581/591 C11-

 MDA Assay Kit Mito-FerroGreen FerroOrange
Target Lipid Peroxide Lipid Peroxidation Lipid Peroxidation Malondialdehyde Ferrous Ion(Fe2+) Ferrous Ion(Fe2+)
Localization Intracellular Mitochondria Intracellular Intracellular Mitochondria Intracellular
Detection
(Fluorescence:Ex/Em)		 Fluorescence
(524 nm/535 nm)		 Fluorescence
(452 nm/470 nm)		 Fluorescence
1. 488 nm/510-550nm
2. 561 nm/600-630nm		 Fluorescence
(540 nm/590 nm)
Colorimetric: 532 nm		 Fluorescence
(505 nm/580 nm)		 Fluorescence
(543 nm/580 nm)
Instrument Fluorescence Microscope,
FCM		 Fluorescence Microscope,
FCM		 Fluorescence Microscope,
FCM,
Microplate Reader		 Microplate Reader Fluorescence Microscope,
Microplate Reader		 Fluorescence Microscope
Sample Live Cell Live Cell Live Cell Cell, Tissue Live Cell Live Cell

Oxidative Stress- and Metabolism-Related Reagents and Kits

Name ROS Assay Kit
-Highly Sensitive DCFH-DA-		 GSSG/GSH Quantification Kit Glutamine Assay Kit-WST Glutamate Assay Kit-WST
Target ROS (Reactive oxygen species) Glutathione (oxidized/reduced) Glutamine Glutamine
Localization Intracellular Intracellular Intracellular/Extracellular Intracellular/Extracellular
Detection
(Fluorescence:Ex/Em)		 Fluorescence
(505 nm/525 nm)		 Colorimetric:412 nm Colorimetric:450 nm Colorimetric:450 nm
Instrument Fluorescence Microscope,
FCM,
Microplate Reader		 Microplate Reader Microplate Reader Microplate Reader
Sample Live Cell Cell, Tissue, Blood Plasma, Red Blood Cell Cell, Culture Medium Cell, Culture Medium

Experimental Example: Evaluating intracellular uptake and redox balance in erastin-induced ferroptosis

 

We investigated the transition of cellular metabolisms in A549 cells treated with erastin, a known ferroptosis inducer. Our results revealed the following.

Results
- The inhibition of cystine uptake by erastin led to a depletion of cysteine, which in turn increased the compensatory uptake of other amino acids.
- Glucose uptake, which typically promotes ferroptosis*, was found to decrease upon erastin treatment, suggesting a potential cellular self-defense mechanism.
- The depletion of cysteine resulted in a decrease in glutathione levels and an increase in Fe2+, ROS, and lipid peroxides, all of which are recognized markers of ferroptosis.

  Cell Line: A549
  Incubation Conditions: 100 μmol/l Erastin/MEM, 37℃, 3h
  *Reference: Xinxin Song, et al., Cell Reports, (2021)

Products in Use
① Amino Acid Uptake: Amino Acid Uptake Assay Kit
② Glucose Uptake: Glucose Uptake Assay Kit-Green
③ Cystine Uptake: Cystine Uptake Assay Kit
④ Intracellular glutathione: GSSG/GSH Quantification Kit
⑤ Intracellular labile Fe: FerroOrange
⑥ Intracellular total ROS: ROS Assay Kit -Highly Sensitive DCFH-DA-
⑦ Lipid Peroxides: Liperfluo

  

Experimental example: Changes in various indicators of cell death induced by drugs

HepG2 cells treated with the apoptosis-inducing agent staurosporine or the ferroptosis-inducing agents Erastin and RSL3. After treatment, extracellular LDH, phosphatidylserine, cell viability, intracellular Fe2+ and lipid peroxidation were determined.
The results showed that apoptosis-induced cells treated with staurosporine showed an increase in phosphatidylserine, a decrease in cell viability and an increase in extracellular LDH, indicating that cell death had occurred. On the other hand, intracellular Fe2+, an indicator of ferroptosis, remained unchanged. In cells treated with Erastin, a ferroptosis inducer, intracellular Fe2+ increased and cell viability decreased, but extracellular LDH and lipid peroxidation (lipid peroxidation: decrease in red fluorescence and increase in green fluorescence) did not increase. In cells in which ferroptosis was more strongly induced by co-treatment with RSL3 in addition to Erastin, increased intracellular Fe2+ and lipid peroxidation were observed. Moreover, decreased cell viability and increased dead cells were detected. Meanwhile, phosphatidylserine showed a lower rate of increase during ferroptosis induction compared to apoptosis-induced cells. These results suggest that cell death can be distinguished by evaluating a combination of cell death indicators.

[Products in use]
Extracellular LDH  : Cytotoxicity LDH Assay Kit-WST (Product code: CK12)
Phosphatidylserine: Annexin V Apoptosis Plate Assay Kit（Product code: AD12）
Cell viability          : Cell Counting Kit-8 (Product code: CK04)
Intracellular Fe2+  : FerroOrange (Product cose: F374) *Normalized with Hoechst 33342 fluorescence intensity
Lipid peroxidation  : Lipid Peroxidation Probe -BDP 581/591 C11- (Product code: L267)

[Experimental conditions]
Cell type: HepG2 cell(2×104 cells/well)
Drugs: Staurosporin(5 μmol/l), Erastin(25 µmol/l), Erastin+RSL3(both 25 µmol/l) *Diluted in serum-free medium

 

 

 

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