The rate at which cells acidify the extracellular medium is frequently used to report glycolytic rate, with the implicit assumption that conversion of uncharged glucose or glycogen to lactate⁻ + H⁺ is the only significant source of acidification. However, another potential source of extracellular protons is the production of CO₂ during substrate oxidation: CO₂ is hydrated to H₂CO₃, which then dissociates to HCO₃⁻ + H⁺.

O₂ consumption and pH were monitored in a popular platform for measuring extracellular acidification (the Seahorse XF Analyzer).

We found that CO₂ produced during respiration caused almost stoichiometric release of H⁺ into the medium. With C2C12 myoblasts given glucose, respiration-derived CO₂ contributed 34% of the total extracellular acidification. When glucose was omitted or replaced by palmitate or pyruvate, this value was 67–100%. Analysis of primary cells, cancer cell lines, stem cell lines, and isolated synaptosomes revealed contributions of CO₂-produced acidification that were usually substantial, ranging from 3% to 100% of the total acidification rate.

Measurement of glycolytic rate using extracellular acidification requires differentiation between respiratory and glycolytic acid production.

The data presented here demonstrate the importance of this correction when extracellular acidification is used for quantitative measurement of glycolytic flux to lactate. We describe a simple way to correct the measured extracellular acidification rate for respiratory acid production, using simultaneous measurement of oxygen consumption rate.

Extracellular acidification is often assumed to result solely from glycolytic lactate production, but respiratory CO₂ also contributes. We demonstrate that extracellular acidification by myoblasts given glucose is 66% glycolytic and 34% respiratory and describe a method to differentiate these sources.