Mitochondrial oxidative
stress assay

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 Rev 94(3); 909-950

Protocol

Mitochondrial oxidative stress assay protocol

Cell Line	HepG2 cell line; other cell types on request
Analysis Platform	Cellomics ArrayScan^® CX7, XTI and VTI (Thermo Scientific)
Test Compound Concentration	8 point dose response curve with top concentration based on 100x C_max or solubility limit*
Compound Requirements	50 µL of a solution to achieve 100x C_max (200x top concentration to maintain 0.5% DMSO) or equivalent amount in solid compound
Number of Replicates	3 replicates per concentration*
Time Points	24 hour*
Quality Controls	Negative control: 0.5% DMSO (vehicle) Positive controls: 2 known mROS-inducing compounds
Data Delivery	Minimum effective concentration (MEC) and AC₅₀ value for each measured parameter, mitochondrial oxidative stress (mROS), and cell health (cell count, nuclear size, DNA structure and cellular ATP)

*Other options available on request.

Related Services
Glucose/galactose mitochondrial toxicity assay Functional mitochondrial toxicity assay (Seahorse) Mitochondrial respiratory complex assay (Seahorse) Mitochondrial biogenesis assay

Data

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.

References

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. Biomolecules 10(1); 93