Rapid analysis of glycolytic and oxidative substrate flux of cancer cells in a microplate

PLoS One. 2014 Oct 31;9(10):e109916. doi: 10.1371/journal.pone.0109916. eCollection 2014.


Cancer cells exhibit remarkable alterations in cellular metabolism, particularly in their nutrient substrate preference. We have devised several experimental methods that rapidly analyze the metabolic substrate flux in cancer cells: glycolysis and the oxidation of major fuel substrates glucose, glutamine, and fatty acids. Using the XF Extracellular Flux analyzer, these methods measure, in real-time, the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of living cells in a microplate as they respond to substrates and metabolic perturbation agents. In proof-of-principle experiments, we analyzed substrate flux and mitochondrial bioenergetics of two human glioblastoma cell lines, SF188s and SF188f, which were derived from the same parental cell line but proliferate at slow and fast rates, respectively. These analyses led to three interesting observations: 1) both cell lines respired effectively with substantial endogenous substrate respiration; 2) SF188f cells underwent a significant shift from glycolytic to oxidative metabolism, along with a high rate of glutamine oxidation relative to SF188s cells; and 3) the mitochondrial proton leak-linked respiration of SF188f cells increased significantly compared to SF188s cells. It is plausible that the proton leak of SF188f cells may play a role in allowing continuous glutamine-fueled anaplerotic TCA cycle flux by partially uncoupling the TCA cycle from oxidative phosphorylation. Taken together, these rapid, sensitive and high-throughput substrate flux analysis methods introduce highly valuable approaches for developing a greater understanding of genetic and epigenetic pathways that regulate cellular metabolism, and the development of therapies that target cancer metabolism.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Proliferation
  • Cell Respiration / physiology
  • Fatty Acids / metabolism
  • Glioblastoma / metabolism*
  • Glucose / metabolism
  • Glutamine / metabolism
  • Glycolysis* / physiology
  • Humans
  • Mitochondria / metabolism*
  • Molecular Biology / instrumentation
  • Molecular Biology / methods*
  • Oxidation-Reduction
  • Oxygen Consumption


  • Fatty Acids
  • Glutamine
  • Glucose

Grant support

This work was conducted in and funded by Seahorse Bioscience. LSPW is an employee and MW was an employee at the time of the work in the company. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.