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Related Experiment Video

Updated: May 30, 2026

A Rapid and Specific Microplate Assay for the Determination of Intra- and Extracellular Ascorbate in Cultured Cells
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A Rapid and Specific Microplate Assay for the Determination of Intra- and Extracellular Ascorbate in Cultured Cells

Published on: April 11, 2014

A high-throughput screening assay of ascorbate in brain samples.

Natalia A Belikova1, Ashley L Glumac, Valentyna Kapralova

  • 1Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, PA 15219, USA.

Journal of Neuroscience Methods
|August 23, 2011
PubMed
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A new fluorescence assay enables rapid measurement of ascorbate (vitamin C) in the central nervous system (CNS). This method accurately quantifies brain ascorbate levels, crucial for understanding redox status and antioxidant defense.

Area of Science:

  • Neuroscience
  • Biochemistry
  • Analytical Chemistry

Background:

  • Ascorbate (vitamin C) is a critical antioxidant in the central nervous system (CNS), influencing redox status and antioxidant capacity.
  • Accurate and efficient measurement of CNS ascorbate is essential for neurological research.
  • Existing methods may lack the speed or specificity required for high-throughput analysis.

Purpose of the Study:

  • To develop and validate a novel, high-throughput fluorescence assay for quantifying ascorbate in brain samples.
  • To establish a reliable method for assessing CNS ascorbate levels in various physiological and pathological conditions.

Main Methods:

  • Development of a fluorescence assay utilizing the reaction between ascorbate and a nitroxide radical probe (AC-TEMPO).

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Last Updated: May 30, 2026

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  • Monitoring the formation of the fluorescent product (AC-TEMPO-H) via fluorescence and electron spin resonance (ESR).
  • Validation of the assay against high-performance liquid chromatography (HPLC) and application to murine tissue samples.

    • Main Results:

    • The assay demonstrated a linear response for ascorbate detection within the micromolar range (0.5-10 microM in the well).
    • The fluorescence signal directly correlated with ascorbate concentration, confirmed by ESR.
    • The assay was successfully validated against HPLC, showing high specificity and reliability.
    • Ascorbate concentrations were measured in murine brain tissue, including samples from traumatic brain injury and hemorrhagic shock models.

    Conclusions:

    • A novel, rapid, and specific fluorescence-based assay for measuring CNS ascorbate has been successfully developed.
    • This high-throughput method provides a valuable tool for assessing brain ascorbate levels in research settings, particularly in studies of neurological injury and disease.
    • The assay's reliability and efficiency facilitate a deeper understanding of the role of ascorbate in CNS health and disease.