Figure 1

Bioenergetic validation of cryopreserved mitochondrial preparations.

(a) Representative oxygen flux recording using closed-chamber, high-resolution respirometry. Rat liver mitochondria (0.25 mg protein ml⁻¹) were maintained under constant stirring. Oxygen consumption (black trace) and oxygen concentration (grey trace) were measured using the Oxygraph 2 K (Oroboros Instruments, Innsbruck, Austria). Sequential additions of reagents were added to assess respiratory states. (b) Mitochondria energised using either glutamate/malate (10 mM/2 mM; black trace) or succinate and rotenone (10 mM/1 μM; grey trace) were treated with oligomycin A (2.5 μg ml⁻¹) and FCCP (0.5 μM titration) to assess leak respiration and spare respiratory capacity respectively. (c) RCR (respiratory control ratio, calculated as the ratio of State 3/State 4 respiration) for freshly isolated mitochondria and trehalose freeze-thawed mitochondria from the same preparation energised using glutamate/malate (10 mM/2 mM). Statistical significance was calculated using a one-way ANOVA corrected for multiple comparisons using Tukey method (NS P > 0.05; GraphPad Prism). (d) RCR for 3 month cryopreserved mitochondria energised using glutamate/malate (10 mM/2 mM). (e) Mitochondria (1 mg protein ml⁻¹) were incubated in the presence of 1 mM ADP and either glutamate/malate (10 mM/2 mM) or succinate/rotenone (10 mM/1 μM) and total ATP content was measured after 45 minutes. Luminescence was measured in the presence and absence of respiratory inhibitors antimycin A (2.5 μM) and oligomycin A (2.5 μg ml⁻¹) to determine background. Data are expressed as means (±s.d.) of at least three independent experiments using mitochondria that had been cryopreserved for between 1 and 3 months. Abbreviations: Mito; mitochondria, Glu; glutamate, Mal; malate, Succ; succinate, FCCP; carbonyl cyanide-p-trifluoromethoxyphenylhydrazone.