# Calculation of ATP production rates using the Seahorse XF Analyzer

## TL;DR

This paper introduces a method to calculate ATP production rates from live cells using the Seahorse XF Analyzer, enabling direct comparison of energy sources like glycolysis and oxidative phosphorylation.

## Contribution

A novel method to calculate ATP production rates using Seahorse XF Analyzer data and empirical conversion factors.

## Key findings

- The method quantifies bioenergetic changes during macrophage polarization and cancer cell adaptation.
- Substantial ATP utilization changes are detected during neuronal depolarization and T cell receptor activation.
- The method provides a single readout for comparing ATP from oxidative phosphorylation and glycolysis.

## Abstract

Oxidative phosphorylation and glycolysis are the dominant ATP‐generating pathways in mammalian metabolism. The balance between these two pathways is often shifted to execute cell‐specific functions in response to stimuli that promote activation, proliferation, or differentiation. However, measurement of these metabolic switches has remained mostly qualitative, making it difficult to discriminate between healthy, physiological changes in energy transduction or compensatory responses due to metabolic dysfunction. We therefore present a broadly applicable method to calculate ATP production rates from oxidative phosphorylation and glycolysis using Seahorse XF Analyzer data and empirical conversion factors. We quantify the bioenergetic changes observed during macrophage polarization as well as cancer cell adaptation to in vitro culture conditions. Additionally, we detect substantive changes in ATP utilization upon neuronal depolarization and T cell receptor activation that are not evident from steady‐state ATP measurements. This method generates a single readout that allows the direct comparison of ATP produced from oxidative phosphorylation and glycolysis in live cells. Additionally, the manuscript provides a framework for tailoring the calculations to specific cell systems or experimental conditions.

This manuscript details a method to calculate ATP production rates using OCR and ECAR data obtained from the Seahorse XF Analyzer. This generates a single readout measuring real‐time rates of ATP produced from oxidative phosphorylation and glycolysis in live cells.