Figure 5

Ca²⁺ retention capacity of freeze-thawed Hela S3 human mitochondria.

Hela S3 mitochondria (0.5 mg protein ml⁻¹) were incubated with Fluo-4FF (0.35 μM) in the presence of succinate (10 mM) and rotenone (1 μM). Pulses of 5 μM CaCl₂ were sequentially added at 3 minute intervals and extra-mitochondrial Fluo-4FF fluorescence measured. Representative raw traces of Ca²⁺ uptake in the presence of (a) ER-000444793 and (b) CsA demonstrate complete Ca²⁺ uptake prior to concentration-dependent Ca²⁺-induced mPT. (c,d) Area under the curve was calculated between 60–660 seconds and results are expressed as % inhibition, normalised to DMSO (0% inhibition) and CsA (5 μM; 100% inhibition). Data are expressed as means (± s.d.) of at least three independent experiments. Curve fitting used a 4-parameter logistic equation (GraphPad Prism). Raw traces are representative of at least three independent experiments. Abbreviations: CsA; cyclosporin A, a.u; arbitrary unit.