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Related Experiment Videos

Quantifying cellular oxidative stress by dichlorofluorescein assay using microplate reader.

H Wang1, J A Joseph

  • 1Neuroscience Laboratory, USDA-ARS, Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University, Boston, MA 02111, USA. wang_us@hnrc.tufts.edu

Free Radical Biology & Medicine
|September 18, 1999
PubMed
Summary
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This study quantifies oxidative stress (OS) in PC12 cells using the dichlorofluorescein (DCF) assay. The assay effectively measures OS induced by various free radical generators, proving useful for evaluating antioxidant efficacy.

Area of Science:

  • Biochemistry
  • Cell Biology
  • Toxicology

Background:

  • Oxidative stress (OS) is linked to aging and degenerative diseases.
  • Quantifying OS in cellular models is crucial for understanding disease mechanisms.
  • PC12 cells provide a relevant model for studying OS.

Purpose of the Study:

  • To establish and validate the dichlorofluorescein (DCF) assay for quantifying OS in PC12 cells.
  • To assess the dose-dependent effects of various pro-oxidants on OS levels.
  • To evaluate the utility of the DCF assay in assessing antioxidant and pro-oxidant activities.

Main Methods:

  • PC12 cells were incubated with various free radical generators for 30 minutes.
  • The dichlorofluorescein (DCF) assay was employed using a fluorescent microplate reader.

Related Experiment Videos

  • Fluorescence intensity was measured to quantify OS induced by hydrogen peroxide (H2O2), AAPH, SIN-1, SNP, and dopamine.

    • Main Results:

    • DCF fluorescence showed a linear correlation with H2O2 and AAPH concentrations (0.1-1 mM).
    • Nonlinear responses were observed with SIN-1, SNP, and dopamine.
    • SIN-1 was the most potent pro-oxidant, increasing fluorescence 70-fold at 100 microM. Dopamine exhibited biphasic effects, acting as an antioxidant at <500 microM and a pro-oxidant at 1 mM.

    Conclusions:

    • The DCF assay, coupled with a fluorescent microplate reader, is a reliable and efficient method for quantifying overall OS in cells.
    • This technique is valuable for evaluating the potency of pro-oxidants and the efficacy of antioxidants.
    • The assay's ability to detect varying responses to different oxidants highlights its versatility in OS research.