Analysis and interpretation of microplate-based oxygen consumption and pH data

Methods Enzymol. 2014;547:309-54. doi: 10.1016/B978-0-12-801415-8.00016-3.


Breakthrough technologies to measure cellular oxygen consumption and proton efflux are reigniting the study of cellular energetics by increasing the scope and pace with which discoveries are made. As we learn the variation in metabolism between cell types is large, it is helpful to continually provide additional perspectives and update our roadmap for data interpretation. In that spirit, this chapter provides the following for those conducting microplate-based oxygen consumption experiments: (i) a description of the standard parameters for measuring respiration in intact cells, (ii) a framework for data analysis and normalization, and (iii) examples of measuring respiration in permeabilized cells to follow up results observed with intact cells. Additionally, rate-based measurements of extracellular pH are increasingly used as a qualitative indicator of glycolytic flux. As a resource to help interpret these measurements, this chapter also provides a detailed accounting of proton production during glucose oxidation in the context of plate-based assays.

Keywords: Bioenergetics; ECAR; Glycolysis; Lactate efflux; Mitochondria; OCR; Permeabilized cells; Proton leak; Respiration; Seahorse XF Analyzer.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Animals
  • Carbon Dioxide / metabolism
  • Cell Respiration
  • Data Interpretation, Statistical
  • Glucose / metabolism
  • Glycolysis
  • Hydrogen-Ion Concentration
  • Hydrolysis
  • Mitochondria / metabolism
  • Molecular Biology / instrumentation
  • Molecular Biology / methods*
  • Oxidation-Reduction
  • Oxygen Consumption / physiology*
  • Permeability
  • Protons
  • Software


  • Protons
  • Carbon Dioxide
  • Adenosine Triphosphate
  • Glucose