ATP-Red enables single-cell bioenergetic phenotyping by flow cytometry

Area of Science:

• * Cellular metabolism and bioenergetics
• * Immunology and infectious disease research
• * Biomedical assay development

Background:

• * Established bioenergetic assays often lack single-cell resolution, potentially obscuring crucial metabolic heterogeneity.
• * Current flow cytometry methods infer cellular energy from protein synthesis, which can be unreliable due to uncoupling from ATP levels.
• * There is a need for precise, single-cell resolution tools to assess cellular energetic states.

Purpose of the Study:

• * To evaluate ATP-Red as a flow cytometry-compatible assay for single-cell bioenergetic phenotyping.
• * To benchmark ATP-Red against established methods for measuring cellular ATP levels and metabolic function.
• * To assess the utility of ATP-Red in capturing bioenergetic heterogeneity in immune cells.

Main Methods:

• * ATP-Red assay was validated using flow cytometry.
• * Benchmarking involved orthogonal approaches: colorimetric ATP quantification, PercevalHR ATP/ADP biosensor, and extracellular flux analysis.
• * Experiments included ATPase inhibition, glycolytic/oxidative blockade, mitochondrial dysfunction, and immune activation models.

Main Results:

• * ATP-Red effectively tracked biologically relevant energetic changes as a relative ATP-linked fluorescence readout.
• * The assay demonstrated high correlation with orthogonal methods across various cellular stress conditions.
• * Combined with immunophenotyping, ATP-Red revealed bioenergetic heterogeneity and pathway utilization in activated T cells and during influenza infection.

Conclusions:

• * ATP-Red provides a practical and scalable solution for single-cell bioenergetic phenotyping.
• * This method overcomes limitations of existing assays by offering single-cell resolution and direct ATP-linked measurement.
• * ATP-Red is valuable for dissecting cellular metabolism in complex biological systems, including immune responses.