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A sensitive flow cytometric method for measuring the oxidative burst

M A Model1, M A KuKuruga, R F Todd

  • 1Division of Hematology/Oncology, 3119 Taubman Center, University of Michigan Medical Center, Ann Arbor 48109-0374, USA.

Journal of Immunological Methods
|March 28, 1997
PubMed
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We developed a new flow cytometry assay to detect the oxidative burst in human neutrophils. This method offers high sensitivity and low nonspecific oxidation for studying neutrophil function.

Area of Science:

  • Immunology
  • Cell Biology
  • Biochemistry

Background:

  • The oxidative burst in neutrophils is a critical component of the innate immune response.
  • Accurate detection of oxidative burst is essential for understanding neutrophil function in various inflammatory and infectious diseases.
  • Existing methods for detecting oxidative burst have limitations in sensitivity, specificity, or ease of use.

Purpose of the Study:

  • To develop and validate a novel flow cytometric method for detecting the oxidative burst in human neutrophils.
  • To compare the sensitivity and specificity of the new method against established assays.

Main Methods:

  • Human neutrophils were covalently labeled on the plasma membrane with 4-carboxydihydrotetramethylrosamine succinimidyl ester.
  • Neutrophil activation was induced using N-formyl-met-leu-phe (FMLP) and phorbol 12-myristate 13-acetate (PMA).

Related Experiment Videos

  • Oxidative burst was measured by an increase in red fluorescence detected by flow cytometry.

    • Main Results:

    • The novel assay demonstrated sensitivity comparable to the cytochrome c oxidation test for FMLP and PMA activation.
    • This method exhibited the lowest nonspecific oxidation and superior sensitivity to FMLP-induced activation compared to other intracellular fluorescent stains.
    • The fluorescence signal remained stable for at least 1.5 hours post-fixation with antioxidant, enabling kinetic studies.

    Conclusions:

    • The developed flow cytometric assay provides a sensitive and reliable method for detecting the oxidative burst in human neutrophils.
    • This technique offers advantages in terms of low nonspecific oxidation and stability, making it suitable for kinetic experiments and various activation stimuli.
    • The assay is a valuable tool for immunological research and clinical diagnostics involving neutrophil function.