Measurement of reactive oxygen species in cultured primary rat hepatocytes

Scope of the method

Alternative method relates to
  • Animal health
Alternative method is situated in
  • Basic Research
Type of alternative method
  • In vitro - Ex vivo
This method makes use of
  • Animal derived cells / tissues / organs
Species from which cells/tissues/organs are derived
Rat
Type of cells/tissues/organs
Hepatocytes

Description

Method keywords
  • Hepatocytes
  • Hepatotoxicity
  • cells
  • rat
  • rodent
  • ROS
  • reactive oxygen species
  • DCFH-DA
Scientific area keywords
  • hepatic toxicity
  • hepatology
  • hepatocytes
  • toxicity testing
  • toxicity
Method description

Basically, the standard operating procedure outlined in this document consists of the following steps, namely, (i) preparation of the cells for the DCFH-DA assay, (ii) DCFH-DA test procedure, (v) processing of the results. Practical details are provided for each of these steps and are followed by some useful tips based upon our own hands-on experience. The fluorescent assay DCFH-DA is based on the ability of the non-fluorescent lypofilic DCFH-DA probe to penetrate viable cells, where it is being deactylated by the presence of intracellular esterases and trapped within the cells. Upon exposure of the cells to a stimulus that generates the oxygen metabolic burst and subsequently the production of ROS, the probe is oxidized and starts to emit an intense green fluorescence. This assay allows the determination of the oxidative stress induction potential of chemical substances. As such, after incubation with the probe, the cells are exposed to the selected substances and the respective emitted fluorescence can be measured for a period of 30 min. The increase in fluorescence is proportional to the amount of ROS that are being formed within the cells. The results can be read by the use of a multiwell scanning fluorimeter (plate reader) using an 485-495nm emission filter and an 520-530nm excitation filter. ROS production is expressed as a ratio of treatment versus control. This method provides a sensitive, reproducible and integrated signal of both attached cells and cells in suspension.

Lab equipment
  • Multiwell scanning fluorimeter (plate reader) using an 485-495nm emission filter and an 520-530nm excitation filter
  • Laminar flow cabinet
  • Thermostated bath
Method status
  • History of use
  • Internally validated

Pros, cons & Future potential

Advantages

The method is sensitive, reproducible and capable of giving an integrated signal of both attached cells and cells in suspension.

Challenges

Unequally seeded cells can give divergent testing results. Take care to handle the seeded cells with caution. Air bubbles in the wells of the 96-well plate can cause negative testing results, gently shake the plate before putting it into the plate reader to avoid this.

Future & Other applications
  • Can be applied to other cell types
  • Possible modification needed

References, associated documents and other information

References

Cadenas S., Aragonés J. and Landázuri M.O. (2010) Mitochondrial reprogramming through cardiac oxygen sensors in ischaemic heart disease. Cardiovascular Research 88(2): 219-228

Burchardt P., Warowicka A., Gozdzicka-Józefiak A. and Wysocki H. (2010) Disturbances of mitochondrial energetic processes and mt-DNA and their role in the etiology of coronary artery disease. Kardiologia Polska 68(8): 947-950

Hamanaka R.B. and Chandel N.S. (2010) Mitochondrial reactive oxygen species regulate cellular signaling and dictate biological outcomes. Trends in Biochemical Sciences 35(9): 505-513

Keston S. and Brandt R. (1965) The fluorometric analysis of ultramicro quantities of hydrogen peroxide. Analytical Biochemistry 11: 1–5

Associated documents
ROS assay.doc

Organisations

Vrije Universiteit Brussel
Pharmaceutical and Pharmacological Sciences (FARM)
In Vitro Toxicology and Dermato-cosmetology
Belgium