Real-Time Assessment of Mitochondrial Function in Cytotrophoblast and Syncytialized Trophoblast Cells Using the Seahorse XFe24 Extracellular Flux Analyzer

Physiological processes utilize variable amounts of energy required for optimal growth, development, and survival. This energy is supplied by total intracellular adenosine triphosphate (ATP) and is mainly generated by mitochondrial oxidative phosphorylation and to a lesser extent via glycolysis. Here, we provide a detailed protocol for obtaining measurements of energy metabolism using the Seahorse XFe24 Extracellular Flux Analyzer. Specifically, this assay measures mitochondrial oxidative phosphorylation based on oxygen consumption rate (OCR) and glycolysis by analyzing the extracellular acidification rate (ECAR) via real-time live cell analysis. Using trophoblast cell lines, this protocol focuses on analyzing mitochondrial respiration for both cytotrophoblasts and syncytiotrophoblasts.