What is ferroptosis?

Diagram of mammalian cell with lipid-reactive oxygen species indicated

Ferroptosis is a non-apoptotic, iron-dependent form of cell death that is distinct from apoptosis or necrosis and involves different biological pathways and physiological processes. This non-apoptotic cell death was first identified in 2003 in a screen of small molecules that selectively kill tumor cells (1) and was formally named ferroptosis in 2012 due to its iron dependence (2). Hallmark traits of ferroptosis include lipid peroxidation and iron accumulation which ultimately result in diminished glutathione peroxidase activity, leading to cell death (3). Ferroptotic cell death is due to the reduction in glutathione peroxidase, causing decreased antioxidant capacity and the accumulation of lipid reactive oxygen species. Several studies have linked ferroptosis with many diseases including ischemia-reperfusion and kidney injuries, nervous system diseases, cancer, and blood diseases.

Several different functional and structural features characterize ferroptosis that are distinct from apoptosis, necrosis, autophagy, and other forms of cell death. Ferroptosis is characterized by a reduction in intracellular glutathione (GSH) and decreased activity of glutathione peroxidase, so that lipid peroxides cannot be oxidized leading to an increase in lipid peroxidation from iron. Structurally, ferroptosis includes mitochondrial changes such as shrinkage, increased membrane density, and loss of cristae. Reagents for detection of oxidative stress, lipid peroxidation, glutathione, and iron, along with reagents for visualizing mitochondrial structure and lipid accumulation can aid in the study of ferroptosis and distinguish this iron-dependent form of cell death from apoptosis and necrosis.

Ferroptosis inducers and inhibitors

The table below shows a selection of known inducers/inhibitors of ferroptosis:

CompoundActionMode of action
L-Buthionine-(S,R)-Sulfoximine (BSO)InducesDepletes GSH by inhibiting γ-glutamylcysteine synthetase, the rate-limiting enzyme for GSH synthesis
ArtemisininInducesIron(II) oxide-reactive endoperoxide that generates ROS
TroloxInhibitsLipophilic antioxidant
Coenzyme Q10 (Cat. No. 457950010 or J65137.06)InhibitsLipophilic antioxidant
IdebenoneInhibitsLipophilic antioxidant
EbselenInhibitsLipophilic antioxidant
Deferoxamine mesylateInhibitsRemoves excess iron, preventing the formation of highly reactive hydroxyl radicals

Probes for assaying ferroptosis, by cellular function

Oxidative stress and reactive oxygen species detection

Ferroptosis is characterized by a general increase in oxidative stress along with iron-dependent accumulation of specific reactive oxygen species (ROS) such as hydroxyl radicals, superoxide, hydroperoxyl radicals, and hydrogen peroxide. This increase in oxidative stress and ROS leads to lipid peroxidation that is a hallmark of ferroptosis and results in cell and tissue damage, particularly in cell membranes. Fluorescent indicators for general intracellular oxidative stress including CellROX reagents and H2DCFDA, and specific ROS indicators for superoxide and hydrogen peroxide enable detection of oxidative stress and the increase in ROS leading to lipid peroxidation and ultimately cell death in ferroptosis.

Lipid peroxidation probes

The accumulation of lipid reactive oxygen species and decreased activity of glutathione peroxidase in ferroptosis leads to an increase in lipid peroxidation from iron. Lipid peroxidation is the oxidative degradation of lipids, resulting in damage to cell membranes. The increase in lipid peroxidation in ferroptosis can be detected through ratiometric fluorescence detection using BODIPY™ lipid probes and with the oxidation of incorporated linoleic acid through a click reaction using the Click-iT™ Lipid Peroxidation Imaging Kit.

Glutathione detection

Intracellular glutathione (GSH), which normally acts as an antioxidant by reduction of reactive oxygen species, is depleted in ferroptosis, resulting in increased oxidative stress and damage. Because GSH is the major free thiol in cells, compounds for detecting thiols including ThiolTracker Violet, monochlorobimane, and monobromobimane can be used to measure the decrease in intracellular GSH levels associated with ferroptosis. Fluorescence and colorimetric microplate assays can also be used to quantitate glutathione levels in cell lysates or other samples.

Iron indicators

Ferroptosis is an iron-dependent process and iron accumulation leads to reduced glutathione peroxidase activity and increased lipid peroxidation through the Fenton reaction. Fluorescent heavy metal indicators such as Phen Green and calcein can be used to detect iron in cells and are quenched in the presence of Fe2+ or Fe3+.

Organelle probes

Various organelles have been implicated in a variety of the ferroptosis pathways including mitochondria (volume reduction, increase in membrane density, and loss of cristae), lysosomes, Golgi, and endoplasmic reticulum. Cell structure probes can be used to investigate changes in organelle structure and function that occur during ferroptosis. See 2021 publication: Organelle-specific regulation of ferroptosis .

Enzymes and metabolites

Enzymes and metabolites involved in ferroptosis, such as glutamate and cholesterol, can be assayed with Amplex Red and Amplex UltraRed based fluorescence microplate assays.

Cellular function

While investigating ferroptosis, it can be useful to examine other aspects of cellular function including cell proliferation, viability, and cell cycle, or to distinguish cell death due to ferroptosis from apoptosis or autophagy.

Primary antibodies for ferroptosis research

ProteinFull namePrimary antibodies
GPX4Glutathione peroxidase 4GPX4 antibodies
ALOX15Arachidonate 15-lipoxygenaseALOX15 antibodies
SLC7A11Solute carrier family 7 member 11SLC7A11 antibodies
BAP1BRCA1 associated protein 1BAP1 antibodies
HSP90Heat shock protein 90HSP90 antibodies
HSPB1Heat shock protein family B (small) member 1HSPB1 antibodies
FANCD2FA complementation group D2FANCD2 antibodies
TP53Tumor protein p53TP53 antibodies
ACSL4Acid coenzyme A ligaseACSL4 antibodies
TFRCTransferrin receptorTFRC antibodies
GLS2Glutaminase 2GLS2 antibodies
DPP4Dipeptidyl peptidase 4DPP4 antibodies
NCOA4Nuclear receptor coactivator 4NCOA4 antibodies
PEBP1Phosphatidylethanolamine binding protein 1PEBP1 antibodies
CARSCysteinyl-tRNA synthetaseCARS antibodies
VDAC2Voltage dependent anion channel 2VDAC2 antibodies
ITGA6Integrin subunit alpha 6ITGA6 antibodies
ITGB4Integrin subunit beta 4ITGB4 antibodies
OTUB1TU deubiquitinase, ubiquitin aldehyde binding 1OTUB1 antibodies
HSPA5Heat shock protein family A (Hsp70) member 5HSPA5 antibodies
FANCD2FA complementation group D2FANCD2 antibodies

Publications citing the use of ferroptosis research reagents



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