Mitochondria consume the majority of cellular oxygen and regulate redox-signalling1.
Toxic drugs can induce mitochondrial reactive oxygen species (mROS) causing mitochondrial/cellular damage which has been linked to the pathology of many diseases2.
The Cyprotex mitochondrial oxidative stress assay detects selective mROS production caused by the toxicity of novel compounds.
ROS trafficking between mitochondria could constitute a positive-feedback mechanism resulting in an elevated production of ROS that could be propagated throughout the cell and may cause perceptible mitochondrial and cellular injury.
1Zorov DB et al. (2014) Physiol Rev94(3); 909-950
Mitochondrial oxidative stress assay protocol
HepG2 cell line; other cell types on request
Cellomics ArrayScan® CX7, XTI and VTI (Thermo Scientific)
Test Compound Concentration
8 point dose response curve with top concentration based on 100x Cmax or solubility limit*
50 µL of a solution to achieve 100x Cmax (200x top concentration to maintain 0.5% DMSO) or equivalent amount in solid compound
Data from Cyprotex's mitochondrial oxidative stress assay
HepG2 cells were plated on tissue culture treated black walled clear bottomed polystyrene plates. The cells were dosed with test compound at a range of concentrations. At the end of the incubation period (24 hours), the cells were labelled with Hoechst (nuclei) and mitoSox® (mROS) then imaged using an automated fluorescent cellular imager, CellInsight CX7 High-Content Screening (HCS) Platform (Thermo Scientific Cellomics).
Figure 1 Representative HCS images of four mROS inducing test compounds alongside vehicle control; nuclei (blue), mROS (green).
Figure 2 Representative dose-response curves for rotenone and chlorpromazine displaying increased mROS formation.
1 Zorov DB et al., (2014) Mitochondrial Reactive Oxygen Species (ROS) and ROS-Induced ROS Release. Physiol Rev 94(3); 909-950 2 JeÅ¾ek P et al., (2020) Redox Signaling from Mitochondria: Signal Propagation and its Targets. Biomolecules10(1); 93
Learn more about toxicology in our popular Mechanisms of Drug-Induced Toxicity guide