Methods for assessing mitochondrial quality control mechanisms and cellular consequences in cell culture

Mitochondrial quality is under surveillance by autophagy, the cell recycling process which degrades and removes damaged mitochondria. Inadequate autophagy results in deterioration in mitochondrial quality, bioenergetic dysfunction, and metabolic stress. Here we describe in an integrated work-flow to assess parameters of mitochondrial morphology, function, mtDNA and protein damage, metabolism and autophagy regulation to provide the framework for a practical assessment of mitochondrial quality. This protocol has been tested with cell cultures, is highly reproducible, and is adaptable to studies when cell numbers are limited, and thus will be of interest to researchers studying diverse physiological and pathological phenomena in which decreased mitochondrial quality is a contributory factor. •We outline methods to assess different facets of the control of mitochondrial health.•We describe biochemical and microscopic methods to assess mitophagy.•We describe methods to measure mtDNA and protein damage.•We describe methods for targeted metabolomics to measure TCA cycle metabolites.•We describe measurement of mitochondrial function in intact and permeabilized cells.