Isolation of Mitochondria from Retinal Pigment Epithelial Cell Cultures and an Application of High-Resolution Respirometric Assay (XFe96 Seahorse Assay)

Abstract

Intracellular Ca²⁺ is strictly regulated to maintain optimal levels for function of cellular organelles as well as mitochondrial respiratory signaling at the tricarboxylic acid cycle and electron transport chain level. Optimal Ca²⁺ concentration for these processes vary between cell types. Furthermore, exposure of mitochondria to sustained, elevated levels of Ca²⁺ induces mitochondrial Ca²⁺ overload and damage to mitochondrial oxidative phosphorylation and ATP production. Isolated mitochondria are widely used to study mitochondrial physiology and drug effects on mitochondrial metabolism and respiratory function. However, isolated mitochondria are easily damaged during the mitochondrial isolation process. The present article describes a mitochondrial isolation method using Ca²⁺-chelation to minimize mitochondrial damage. We follow up the isolation process with an application that requires an optimized buffer Ca²⁺ concentration: the characterization of their respiratory function using a high-resolution respirometric assay.

Keywords: Calcium chelator; Mitochondrial isolation; Organelle isolation.