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

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.