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ADME PK

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 Cmax or solubility limit*
Compound Requirements 50 µL of a solution to achieve 100x Cmax (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 Ježek P et al., (2020) Redox Signaling from Mitochondria: Signal Propagation and its Targets. Biomolecules 10(1); 93

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