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Updated: May 25, 2026
Bioenergetics and the Oxidative Burst: Protocols for the Isolation and Evaluation of Human Leukocytes and Platelets
Published on: March 27, 2014
Measurement of oxidative burst in neutrophils
1Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.
Abstract:
Polymorphonuclear neutrophils (PMNs) generate reactive oxygen species (ROS) during phagocytosis and in response to soluble agonists. This functional response, termed oxidative burst, contributes to host defense, but it can also result in collateral damage of host tissues. To study this important PMN response, different methods have been developed that are based on the assessment of oxidative burst by measuring intracellular ROS production or formation of ROS in the extracellular space. Among the different methods that were developed, the following two are particularly widely used because of their convenience and accuracy. The first method depends on the reduction of cytochrome c, which can be assessed by photometry, while the second method relies on changes in the fluorescence properties of dihydrorhodamine 123, which can be assessed by flow cytometry.
Insights
Polymorphonuclear neutrophils (PMNs) generate reactive oxygen species (ROS) during an oxidative burst, crucial for host defense but potentially damaging. Two accurate and convenient methods, cytochrome c reduction and dihydrorhodamine 123 fluorescence, are widely used to study this response.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Polymorphonuclear neutrophils (PMNs) are key immune cells involved in host defense.
- PMNs generate reactive oxygen species (ROS) through an oxidative burst during phagocytosis and in response to agonists.
- While essential for pathogen clearance, excessive ROS production can lead to host tissue damage.
Purpose of the Study:
- To review and highlight commonly used methods for assessing the oxidative burst in PMNs.
- To provide an overview of techniques that measure intracellular and extracellular ROS production.
- To emphasize the importance of studying PMN oxidative burst for understanding host defense and pathology.
Main Methods:
- Assessment of oxidative burst by measuring intracellular ROS production.
- Assessment of oxidative burst by measuring extracellular ROS formation.
- Specific methods discussed include cytochrome c reduction (photometry) and dihydrorhodamine 123 fluorescence (flow cytometry).
Main Results:
- The cytochrome c reduction assay quantifies ROS by measuring the reduction of cytochrome c, typically via photometry.
- The dihydrorhodamine 123 assay measures ROS by detecting changes in fluorescence properties, commonly analyzed by flow cytometry.
- Both methods are recognized for their convenience and accuracy in studying PMN oxidative burst.
Conclusions:
- Accurate measurement of PMN oxidative burst is critical for understanding immune responses.
- Cytochrome c reduction and dihydrorhodamine 123 fluorescence are widely adopted, reliable techniques for ROS detection.
- These methods facilitate research into the dual role of oxidative burst in host defense and inflammatory diseases.
