Figure 2 | Scientific Reports

Figure 2

From: Identification of ER-000444793, a Cyclophilin D-independent inhibitor of mitochondrial permeability transition, using a high-throughput screen in cryopreserved mitochondria

Figure 2

Validation of freeze-thawed mitochondria using TMRM.

Rat liver mitochondria (1 mg protein ml−1) were incubated with TMRM (2 μM) in the presence of respiratory substrates and compounds: (a) FCCP; (b) rotenone ± oligomycin A (2.5 μg ml−1) and (c) myxothiazol. Mitochondria were exposed to mitochondrial toxins in a 3-fold dilution series and TMRM fluorescence (ex. 560/em. 590 nm) measured after 10 minute incubation. (d) FCCP concentration-response in freshly isolated mitochondria and trehalose freeze-thawed mitochondria from the same preparation. Results are expressed as percentage (%) depolarisation, normalised to DMSO (0% depolarisation) and antimycin A (2.5 μM) plus oligomycin A (2.5 μg ml−1; 100% depolarisation). 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. Curve fitting used a 4-parameter logistic equation (GraphPad Prism). Abbreviations: FCCP; carbonyl cyanide-p-trifluoromethoxyphenylhydrazone.

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