Cells previously transfected with siRNAs for 72 to 96 hours were seeded onto 35-mm glass bottom microwell dishes (MatTek Corporation), infected with mtPericam adenovirus and incubated for 48 hours. Pericam fluorescence was determined in cells in Tyrode’s solution [140 mM NaCl, 10 mM glucose, 5.4 mM KCl, 1.8 mM CaCl2, 2.0 mM MgCl2, 1.2 mM KH2PO4, 5 mM Hepes (pH 7.4) with NaOH). Imaging was performed at room temperature with a Leica TCS SP8 STED confocal microscope. Pericam was excited at 405 and 480 nm, and its emission was recorded at 535 nm. ATP (100 μM) or PDGF (20 ng/ml) was added by micropipet (in amounts of 10 μl) to trigger mitochondrial Ca2+ uptake. Recordings were performed every 5 s for at least 10 min. mtPericam signals were quantified by ImageJ. The rise in amplitude above baseline and the AUC for 5 min after PDGF application were calculated. Peak amplitude (R) was calculated using RpeakRbaseline. The AUC was determined by subtracting the AUC at the baseline ratio. Summary data represent the average difference in basal and peak mitochondrial [Ca2+].

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