Ferroptosis Research Solutions

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.

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

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.

• Generalized oxidative stress
• Selective ROS detection
• Fluorescent protein–based redox sensors
• Microplate assays for ROS

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.

• Lipid peroxidation detection
• Ratiometric lipid peroxidation detection (Cat. No. D3861; Cat. No. B3932)
• Lipid-peroxidation derived protein modification 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.

• Intracellular glutathione levels
• Quantitative fluorescence glutathione measurement (Cat. No. M50550; Cat. No. EIAGSHF)
• Quantitative colorimetric glutathione measurement

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⁺.

• Indicators for iron
• Ion and heavy metal indicators

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 .

• Cell structure probes

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

• Glutamate and cholesterol assays

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.

• Cell function assays
• Apoptosis assays
• Autophagy assays
• Cell viability assays

1. Cell Death Dis. 2019 Oct; 10(10): 755. Reagents used: BODIPY 581/591 C11 (Lipid Peroxidation Sensor)
2. Blood. 2020 Sep 10; 136(11): 1303–1316. Reagents used: BODIPY 665/676 (Lipid Peroxidation Sensor); CellROX Deep Red Flow Cytometry Assay Kit
3. Autophagy. 2021; 17(12): 4266–4285. Reagents used: BODIPY 581/591 C11 (Lipid Peroxidation Sensor); Phen Green SK, Diacetate
4. Redox Biol. 2020 Feb; 30: 101417. Reagents used: Image-iT Lipid Peroxidation Kit, for live cell analysis; CellEvent Caspase 3/7 detection reagent

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2. Dixon SJ et al. (2012) Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell 149:1060–1072.
3. Li J, Cao F, Yin Hl et al. (2020) Ferroptosis: past, present and future. Cell Death Dis 11:88.