When measuring mitochondrial function or the effects of an unknown on mitochondrial function, it can be difficult to determine a mechanism using whole organisms or tissue. When this is the case, isolated mitochondria provide a simple and biochemically relevant experimental model. Cayman’s MitoCheck® Mitochondrial (Tissue) Isolation Kit includes a complete package of reagents and step-by-step instructions for isolating coupled mitochondria* from freshly harvested mammalian heart, liver, or kidney tissue through a process of differential centrifugation in isotonic buffers. *Coupled mitochondria are defined as mitochondria capable of phosphorylating ADP at the F1FO-ATP synthase (Complex V) through the utilization of a proton (H+) gradient, which is generated by the electron transport chain (ETC) in the presence of appropriate substrates (e.g., succinate) and in the absence of uncouplers and mitochondrial inhibitors.