Substrates, co-substrates, and additives (stock concentration)	Final concentration	Volume to add to 1 mL assay media
ADP	ADP (0.5M), pH 7.2 in H2O	4 mM	8 μL
Fatty-acid free BSA	BSA [10% (w/v)] in H2O	0.2% (w/v)	20 μL
rPFO	rPFO; commercially XF PMP (10 μM)	1–3 nM	0.1–0.3 μL
NADH-linked (Complex I) substrates	Pyruvate (0.5M), pH 7.2 in H2O Malate (0.5M), pH 7.2 in H2O	5 mM 0.5 mM	10 μL 1 μL
	Glutamate (0.5M), pH 7.2 in H2O Malate (0.5M), pH 7.2 in H2O	5 mM 5 mM	10 μL 10 μL
	Palmitoyl CoA (10 mM) in H2O Malate (0.5M) in H2O Carnitine (0.5M) in H2O	40 μM 0.5 mM 0.5 mM	4 μL 1 μL 1 μL
	Palmitoyl carnitine (10 mM) in EtOH Malate (0.5M)	40 μM 0.5 mM	4 μL 1 μL
Q-linked (Complex II or III) substrates	Succinate (0.5M), pH 7.2 in H2O Rotenone (2 mM) in EtOH	5 mM 2 μM	10 μL 1 μL
	Glycerol-3-phosphate (0.25M), pH 7.2 in H2O Rotenone (2 mM) in EtOH CaCl2 (1M) in H2O	5 mM 2 μM 700 μM total	20 μL 1 μL 0.7 μL
Complex IV substrates	Ascorbate (1M), pH 7.2 in H2O TMPD (10 mM) Antimycin A (2 mM)	10 mM 100 μM 1 μM	10 μL 1 μL 0.5 μL