Table 1.

Measurement of mitochondrial proton leak

Methods	Mechanism	Advantages	Limitations	Ref.
Clark-type oxygen electrode	[O2] change	Can measure phosphorylation related O2 consumption in one experiment.	One sample each time. Needs isolated mitochondria or permeabilized cells. Requires ATPase blocker. Cannot exclude “proton slip”.	[12]
Seahorse assay	[O2] change	Can measure phosphorylation related O2 consumption, maximal respiration in one experiment. Good for isolated mitochondria or intact cells. Suitable for high throughput (24- or 96-well plates). Measure ECAR simultaneously.	Requires ATPase blocker. Cannot exclude “proton slip”.	https://www.agilent.com/en/product/cell-analysis/how-to-run-an-assay.
Mitoplast patch-clamp	Proton electricity current	Direct Accurate	Requires mitoplast. Requires high skills.	[15, 16]
pH indicator-based measurement	pH indicator fluorescence change	Direct No substrate. Reflects the purely proton leak. Suitable for high throughput.	Requires a genetically encoded pH indicator.	[20]