Ferroptosis Assay

Ferroptosis is a regulated cell death process triggered by the toxic accumulation of lipid peroxides on cell membranes. This iron-dependent form of cell death distinguishes itself from other mechanisms, like apoptosis. Lipid peroxides, products of routine metabolic processes, induce oxidative damage to cell membranes, contributing to the distinctive features of ferroptosis.

Figure 1. Mechanisms governing ferroptosis and reversing chemotherapy resistance. Three pathways initiate the process of ferroptosis and chemotherapy resistance reversal: the canonical GPX4-regulated pathway, iron metabolism pathway and lipid metabolism pathway.^[1]

One extensively researched aspect of ferroptosis is its involvement in cancer. Ferroptosis exhibits dual effects, acting both pro-tumor and anti-tumor. Ferroptosis can promote tumor growth by causing the death of immune cells that would normally hinder cancer progression. Additionally, the heightened production of reactive oxygen species (ROS) during ferroptosis can also stimulate tumor cell proliferation. However, cancer cells are vulnerable to ferroptosis due to their elevated levels of iron and ROS, resulting from their increased metabolic activity for uncontrolled cell growth. Numerous studies have demonstrated that inducing ferroptosis in cancer cells can be a successful method for selectively eliminating them, while minimizing harm to surrounding healthy cells.

Creative Bioarray offers a comprehensive ferroptosis assay, a cutting-edge solution for exploring and understanding the intricate mechanisms of cell death. Our assay provides a sophisticated platform to investigate ferroptosis induction and its impact on various cell types.

Detecting Hallmarks of Ferroptosis

• Lipid Peroxidation Assays:
  Malondialdehyde (MDA) Assay: MDA is a byproduct of lipid peroxidation. Assays such as the thiobarbituric acid reactive substances (TBARS) assay can measure MDA levels.
  BODIPY™ 581/591 C11 Assay: This dye changes its fluorescence emission spectrum upon oxidation, making it useful for detecting lipid peroxidation.
• Glutathione (GSH) Measurement:
  Total Glutathione Assay: Ferroptosis is associated with a decrease in intracellular GSH levels. Colorimetric or fluorometric assays can measure total GSH content.
  GSSG/GSH Ratio Measurement: An increase in the ratio of oxidized glutathione (GSSG) to reduced glutathione (GSH) is indicative of oxidative stress.
• Iron Measurement:
  Iron Staining: Prussian blue staining or other iron-specific dyes can be used to visualize intracellular iron accumulation.
• ROS Detection:
  Dihydroethidium (DHE) Staining: DHE is a fluorescent probe that detects superoxide radicals. Increased ROS production is a hallmark of ferroptosis.
  CellROX™ Green Reagent: This dye is used to measure oxidative stress in live cells and is often used to assess ROS levels.
• Mitochondrial Membrane Potential (Δψm):
  Tetramethylrhodamine Methyl Ester (TMRM) Staining: TMRM is a cationic dye that accumulates in active mitochondria. A decrease in mitochondrial membrane potential is associated with ferroptosis.
• Cell Viability and Cytotoxicity Assays:
  MTT Assay: Measures cell metabolic activity, and a decrease in MTT reduction can indicate cell death.
  LDH Release Assay: Measures the release of lactate dehydrogenase, a marker of cell membrane damage.
• Genetic Markers:
  GPX4 Expression: Ferroptosis is often associated with decreased expression of the antioxidant enzyme glutathione peroxidase 4 (GPX4).
  System Xc- Activity: System Xc- is a cystine/glutamate antiporter, and inhibition of its activity is linked to ferroptosis.
• Morphological Changes:
  Transmission Electron Microscopy (TEM): TEM can reveal characteristic morphological changes in cells undergoing ferroptosis, such as mitochondrial shrinkage and increased membrane density.

References:

1. Zhang, Chen et al. "Ferroptosis in cancer therapy: a novel approach to reversing drug resistance." Molecular cancer vol. 21,1 47. 12 Feb. 2022, doi:10.1186/s12943-022-01530-y

2. Li, Jie et al. "Ferroptosis: past, present and future." Cell death & disease vol. 11,2 88. 3 Feb. 2020, doi:10.1038/s41419-020-2298-2

3. Murray MB, Leak LB, Lee WC, Dixon SJ. Protocol for detection of ferroptosis in cultured cells [published online ahead of print, 2023 Aug 8]. STAR Protoc. 2023;4(3):102457. doi:10.1016/j.xpro.2023.102457