Which protocol should I use for the Seahorse Bioscience XF96 Extracellular Flux Analyzer and your cardiomyocytes?

The XF96 Extracellular Flux Analyzer (Seahorse Bioscience) is a non-invasive, label-free, high-throughput instrument that measures metabolic activity of living cells by simultaneously monitoring mitochondrial respiration and glycolysis. Our Application Protocol describes how you can culture iCell® Cardiomyocytes by directly plating into an XF96 Cell Culture Microplate and provides basic instructions for bioenergetics data acquisition and analysis for assessing in vitro mitochondrial function and therapeutic intervention potential.

General protocol recommendations:

  • Assay timing: 7 days post-plating using a direct-plate method
  • Assay density: 15,000 plated cells in 80 μl per well for the XF96 microplate, using only the 60 inner wells
    See our User’s Guide for instructions on calculating the target plating density from plating efficiency.
  • Assay medium: XF Assay Medium + 10 mM galactose, 1 mM pyruvate, 4 mM glutamine
  • Assay extracellular matrix (ECM): 0.1% gelatin


  1. Application Protocol: Performing Bioenergetic Analysis:XF96 Extracellular Flux Analyzer
  2. Rana et al. (2012) Characterization of Human-Induced Pluripotent Stem Cell-Derived  Cardiomyocytes: Bioenergetics and Utilization in Safety Screening.
  3. Poster Presentation: Fiene et al. (2012 CDI UGM) Human iPSC-Derived Cells for Modelling Cellular Bioenergetics: Building a Metabolic Profile Using the XF Mito Stress Test