Assessing mitochondrial respiratory bioenergetics in whole cells and isolated organelles by microplate respirometry

Mitochondria are responsible for the generation of ATP by oxidative phosphorylation; however, these multifaceted organelles regulate many other key cellular functions as well, such as calcium homeostasis, apoptosis, and biosynthesis of steroid hormones, heme and phospholipids. In order to carry out these functions, mitochondria establish physical and functional connections with other organelles such as the plasma membrane, lipid droplets/vesicles, peroxisomes, endosomes, and the endoplasmic reticulum. Dysregulation of any of the aforementioned processes or inter-organelle contacts can lead to mitochondrial dysfunction and subsequent changes in oxygen consumption and ATP production. Seahorse technology has become a critical tool for quantification of mitochondrial oxygen consumption and can help differentiate primary mitochondrial disorders from disorders where alterations in mitochondrial metabolism are consequences of a prior, upstream insult. In this chapter, we describe the application of Seahorse technology for assaying mitochondrial respiration in whole cells, permeabilized cells and isolated mitochondria. We leave it to the researcher's discretion to determine which of these approaches will generate the most physiologically relevant data based on the model system and research question at hand.