Ferroptosis: Mechanisms in Disease and Kit Selection

Science Note

Lipid Droplet Biology: Emerging Disease Links [Nov. 11, 2025] 

Previous Science Note

Lipid droplets are active organelles that not only store fatty acids but also modulate redox signaling and intersect with cell death pathways. Their composition and turnover shape susceptibility to lipid peroxidation and the inflammatory state of the aging brain, linking droplet biology to ferroptosis and Alzheimer’s disease. Therefore, understanding lipid-droplet biology is important for research in cancer and neurological disorders. Recent work shows that neutral lipids inside droplets can undergo peroxidation and that an FSP1–CoQ10 pathway localized to droplets restrains this process. Another study indicates that Aβ drives DGAT2-dependent triacylglycerol synthesis in microglia, increasing lipid droplets and diminishing Aβ clearance.

1. FSP1-mediated lipid droplet quality control prevents neutral lipid peroxidation and ferroptosis (Nature Cell Biology, 2025)
Summary: This study shows that lipid droplets are not passive storage and that their neutral lipids can undergo peroxidation. It further demonstrates that FSP1 on the lipid droplet membrane regenerates CoQ10 to its antioxidant form and halts peroxidation inside the droplet, defining a lipid droplet specific lipid quality control pathway and revealing a new defense layer in which FSP1 suppresses ferroptosis that begins in lipid droplets under lipid droplet rich conditions.

Highlighted technique: The study visualizes lipid peroxidation at intracellular lipid droplets by combining a lipid peroxidation fluorescent probe with a droplet selective lipid droplet dye in live cell imaging. Peroxidation within droplets increased with the FSP1 inhibitor FSEN1 and was suppressed by the radical trapping antioxidant ferrostatin, supporting localized lipid peroxidation at lipid droplets.

 Related technique   Lipid Droplet Detection (as used in this article), Lipid Peroxidation Detection

2. Amyloid-β induces lipid droplet-mediated microglial dysfunction via the enzyme DGAT2 in Alzheimer’s disease (Immunity, 2025)
Summary: Building on prior observations that microglia near Aβ plaques harbor more lipid droplets and show reduced phagocytosis, this study shows that Aβ exposure promotes DGAT2-dependent triacylglycerol synthesis in microglia, leading to lipid-droplet accumulation. Inhibiting DGAT2, the endoplasmic reticulum enzyme that catalyzes the final step from DAG to TAG, reduces droplets, restores microglial Aβ uptake, and lowers plaque burden in the Alzheimer’s mouse model.

Highlighted technique: This study used microglia isolated from the mouse brain and assayed phagocytosis with Aβ conjugated to a pH-sensitive fluorogenic probe that becomes fluorescent in acidic compartments. Upon staining lipid droplets with fluorescent dyes, microglia from an AD model mouse contained more lipid droplets and showed reduced Aβ uptake compared with wild-type.

 Related technique   Lipid Droplet Detection, pH Sensor Labeling

Ferroptosis Indicators (click to open/close)
Target Kit & Probes
Lipid Droplet Staining Lipi-Blue/ Green/ Red/ Deep Red
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-
Malondialdehyde Detection MDA Assay Kit
Mitochondrial superoxide detection MitoBright ROS Deep Red - Mitochondrial Superoxide Detection
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
Application Note (click to open/close)
  > Propranolol-Induced Lipid Accumulation in HepG2 Cells

We treated HepG2 cells with Propranolol, which is known to cause lipid accumulation in hepatocytes. Using high-content fluorescence imaging, we were able to visually confirm and quantify the increase in lipid droplets within the cells.
By analyzing the captured images, we measured: The number of lipid droplets / Their size (area) / Overall fluorescence signal. This workflow provides a clear, quantitative readout of drug-induced hepatotoxicity.

Lipid droplet imaging


HepG2 cells were treated with propranolol 0, 10, or 30 μmol/l, lipid droplets were stained with Lipi-Green and nuclei with Hoechst 33342 and observed using a fluorescence microscope (Ti2-E inverted microscope). The results showed that lipid droplets increased in a propranolol concentration-dependent manner.

Nucleus (blue: Hoechst33342 ): Ex 385 nm, Em 460 nm
Fat droplet (green: Lipi-Green): Ex 475 nm, Em 535nm

Analysis of fat droplet accumulation relative to drug treatment concentration

High Content Analysis (HCA) microscope system
(Nikon Corporation https://www.microscope.healthcare.nikon.com/)

For details of staining and analysis methods, please refer to "APPLICATION NOTE: Hepatotoxicity test of drug-induced lipidosis using high-content imaging" by Nikon Corporation.

 From the fluorescence images obtained, the accumulation of lipid droplets per cell was analyzed by measuring cell number from nuclei and area, number, and fluorescence intensity from lipid droplets. The results showed that the number and area of lipid droplets increased in a propranolol concentration-dependent manner, with lipid droplets forming significantly under concentration conditions of 20 μmol/l or higher. The DS-Qi2 camera, which can capture a wide range of cellular areas in a single shot, was used for imaging, and the EDF module of NIS-Elements software, which can acquire focused images of all lipid droplets, was used for analysis, enabling quantitative analysis with highly reliable statistical data. The EDF module of the NIS-Elements software allows for the acquisition of focused images of all lipid droplets.
   
 

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 an Overview Map!
      
(Click to open)

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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