Measuring Mitochondrial Calcium Load

The CLC of frozen HEK293F-derived mitochondria was determined using a method based on that previously described for fresh rat liver mitochondria (23 (link)). Briefly, a buffer (130 mM Sucrose, 37.54 mM KCl, 2.5 mM KH₂PO₄, 5 mM HEPES) containing 0.5 mM Ca²⁺ and supplemented with 15 mM succinate, 2.5 μM rotenone and 0.15 μg/ml cell impermeant Fluo 5N (Life Technologies), was added to each well of a 384 well microplate (control wells also contained 0.24 μl/well DMSO or 3 μM cyclosporine). Thawed mitochondria suspended at 0.6 mg/ml were added to all wells to initiate the assay, and the fluorescence measured every 5 minutes at 520 nm in a PHERAstar FS microplate reader (BMG LabTech, Aylesbury, UK), (25 reads over 2 hours). Area under the curve (AUC) values for the first 10 reads were exported and the concentration-response relationship was modelled using the following formula: y=((B-A)/(1+(10^X/10^C)^D)+A where B=max, A=min, C=IC50, D=slope. The fitted curves were then used to calculate a pXC₅₀ value.