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SALSA: a novel flow cytometry assay to detect ascorbate at the single-cell level.

Bella Lee1, Wendy Wan-Ting Chou2, Heng-Yi Chen2

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Redox Biology
|August 25, 2025
PubMed
Summary

We developed SALSA, a new flow cytometry method to measure intracellular ascorbate (AA) in single cells. SALSA uses a fluorescent probe to detect AA levels, enabling new insights into redox biology and immunology.

Keywords:
AscorbateVitamin C

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Area of Science:

  • Biochemistry
  • Cell Biology
  • Immunology

Background:

  • Ascorbate (AA) is vital for antioxidant defense, enzymatic functions, and immune responses.
  • Accurate single-cell measurement of intracellular AA has been a significant technical hurdle.
  • Emerging roles of AA in epigenetic regulation and redox biology necessitate precise quantification methods.

Purpose of the Study:

  • To develop a sensitive and specific method for single-cell intracellular ascorbate (AA) quantification.
  • To characterize the dynamics of AA in immune cells and identify its transporters.
  • To establish a robust assay for probing AA in various biological contexts.

Main Methods:

  • Development of the Single-cell Ascorbate Level Sensing Assay (SALSA) using flow cytometry.
  • Utilized 4,5-diaminofluorescein (DAF-2) as a selective AA reporter, detecting a red-shifted fluorescent adduct.
  • Employed CRISPR technology in conjunction with SALSA to identify AA transporters.

Main Results:

  • SALSA enables sensitive, specific, and quantitative detection of intracellular AA at the single-cell level.
  • Identified SVCT2 as the primary ascorbate transporter in a human cell line model using SALSA and CRISPR.
  • Revealed significant heterogeneity in AA levels across different immune cell subsets and developmental stages.

Conclusions:

  • SALSA is a robust and accessible method for single-cell resolution of AA dynamics.
  • The assay has broad applicability in redox biology, immunology, and metabolic research.
  • SALSA facilitates the study of AA's role in cellular functions and disease states.