Table 3: Defining respiratory parameters for intact cells and three-dimensional structures.

Plate-based oxygen consumption measurements have helped standardize respiratory parameters for intact cells, though the broad experimental framework had been established decades prior. It is almost always most informative to report raw, quantitative rates. Nonetheless, internally normalized parameters can be useful when it is difficult to control for cell number or biomass, such as when working with tissue pieces. This scaling is independent of cell number or sample size, and allows comparisons across different laboratories, experimental platforms, and model systems. Further definitions and interpretations of these parameters, as well as guidelines for calculation, have been previously published^9,12,16. Specific points are discussed further in the main text.

Term	Description of respiratory parameters in intact cells or 3D structures
Basal (or resting/initial) respiration	The initial respiratory rate in intact cells or multicellular structures largely reflects the resting ATP demand. In proliferating cells, a substantial portion of this reflects the energetic costs of biosynthesis and cell division. In differentiated cells, the initial rate may be quite low without activation from external, physiologically relevant stimuli. In most cells, roughly 80% of the basal respiratory rate is coupled to ATP synthesis, with the remainder attributable to processes that use the mitochondrial membrane but do not generate ATP66.
Proton leak	Oxygen consumption is not completely coupled to ATP synthesis, as a residual respiratory rate persists in the presence of oligomycin. This rate reflects a composite of processes that consume the membrane potential despite ATP synthase inhibition. Changes in proton leak-linked respiration may indicate altered energy expenditure, and can be substantial upon activation of brown adipocytes.
Uncoupler-stimulated (or maximal) respiration	As basal respiratory rates are restrained by the ATP demand of the cell, they often do not accurately reflect the ability of a cell to respond to increased energy requirements. Addition of a titrated amount of protonophore, however, decouples (or ‘uncouples’) respiratory chain activity from cellular ATP requirements. The rate estimates the maximal capacity of mitochondria to transport and oxidize energy substrates.
Non-mitochondrial oxygen consumption	In microplate-based platforms, mitochondrial respiration should always be corrected by subtracting the rate insensitive to respiratory chain inhibition, usually with the complex I inhibitor rotenone and the complex III inhibitor antimycin A. Apart from experimental conditions where activation of non-mitochondrial oxidases is expected, a substantial component of this rate in the XF Analyzer may be instrument background.
	Internally scaled parameters independent of cell number or sample size
Spare/reserve respiratory capacity	The spare respiratory capacity is often calculated as the absolute difference between the basal and uncoupler-stimulated rates of respiration. It can also be presented as a ratio-based parameter (i.e. maximal rate:basal rate) as an internally normalized parameter for the relative ability of cells or 3D structures to respond to an increased energy demand.
Coupling efficiency & other measures	The fraction of the basal respiratory rate that is coupled to ATP synthesis (i.e. oligomycin-sensitive respiration:basal respiration) can be represented as a percentage to allow for comparison across model systems. In most cell types this is value is around 80%. An additional, internally normalized metric is the ratio of uncoupler-stimulated respiration to the proton leak-linked respiration, sometimes called the ‘cell respiratory control ratio17.’