Free cytosolic Ca2+ concentration was monitored ratiometrically as ratio signals at 340 nm and 380 nm excitation and 510 nm emission using fura-2 (ref. 23 (link)).
To monitor free mitochondrial Ca2+ concentration, cells were cotransfected with mitochondria-targeted ratiometric-pericam17 (link) and measured using a fluorescence microscope (Eclipse TE300, Nikon, Vienna, Austria) as previously described2 (link),24 (link). Due to the properties of the sensor, data presented are normalized to 1–(F433/F0) and non-normalized values are shown in theSupplementary Information, Table S1 .
Mitochondrial Ca2+ uptake from the medium was measured with Calcium Green-5N (0.1 μM; Molecular Probes Europe, Leiden, The Netherlands) according to previously described methods25 (link). Isolated mitochondria (0.5 mg protein) were resuspended in 2 ml high KCl-buffer (110 mM KCl, 0.5 mM KH2PO4, 1 mM MgCl2, 20 mM HEPES, 0.01 mM EGTA, 5 mM succinate, 0.004 mM rotenone at pH 7.2). Fluorescence was recorded at room temperature at 506 nm excitation and 532 nm emission (Hitachi F-4500; Inula, Vienna, Austria). Fmin and Fmax were established by addition of 1 mM EGTA and 10 mM Ca2+. The KD of Calcium Green-5N under the respective experimental conditions was 7.54 (6.62–8.59) μM.
Measurement of mitochondrial Ca2+ uptake in digitonin-permeabilized HeLa cells was performed in high KCl-buffer containing 1 μM thapsigargin and 2.5 × 106 cells per ml using Calcium Green-5N as described above. The plasma membranes were permeabilized by treatment with 3 μM digitonin for 6 min.
Ca2+ measurements in isolated single mitochondria were performed to previously described methods26 (link). A 20 μl drop of the suspension of fura-2-loaded mitochondria was placed on a coverslip and mounted onto the fluorescence microscope (Axiovert 200M; Zeiss, Vienna, Austria). After 8 min, attached mitochondria were perfused with assay buffer (20 mM HEPES, 130 mM KCl, 1 mM MgCl2, 0.5 mM KH2PO4, 10 mM succinate, 2 mM malate, 0.5 mM EGTA, 0.6 mM ATP, 0.05 mM ADP at pH 7.2) at 2 ml per min. Ca2+ uptake into single mitochondria was monitored on changing the perfusion solution from Ca2+ free buffer to a buffer containing 10 μM free Ca2+.
Free Ca2+ concentration within the lumen of the endoplasmic reticulum Ca2+ was measured with D1 (ref. 27 (link)), as described previously28 (link).
To monitor free mitochondrial Ca2+ concentration, cells were cotransfected with mitochondria-targeted ratiometric-pericam17 (link) and measured using a fluorescence microscope (Eclipse TE300, Nikon, Vienna, Austria) as previously described2 (link),24 (link). Due to the properties of the sensor, data presented are normalized to 1–(F433/F0) and non-normalized values are shown in the
Mitochondrial Ca2+ uptake from the medium was measured with Calcium Green-5N (0.1 μM; Molecular Probes Europe, Leiden, The Netherlands) according to previously described methods25 (link). Isolated mitochondria (0.5 mg protein) were resuspended in 2 ml high KCl-buffer (110 mM KCl, 0.5 mM KH2PO4, 1 mM MgCl2, 20 mM HEPES, 0.01 mM EGTA, 5 mM succinate, 0.004 mM rotenone at pH 7.2). Fluorescence was recorded at room temperature at 506 nm excitation and 532 nm emission (Hitachi F-4500; Inula, Vienna, Austria). Fmin and Fmax were established by addition of 1 mM EGTA and 10 mM Ca2+. The KD of Calcium Green-5N under the respective experimental conditions was 7.54 (6.62–8.59) μM.
Measurement of mitochondrial Ca2+ uptake in digitonin-permeabilized HeLa cells was performed in high KCl-buffer containing 1 μM thapsigargin and 2.5 × 106 cells per ml using Calcium Green-5N as described above. The plasma membranes were permeabilized by treatment with 3 μM digitonin for 6 min.
Ca2+ measurements in isolated single mitochondria were performed to previously described methods26 (link). A 20 μl drop of the suspension of fura-2-loaded mitochondria was placed on a coverslip and mounted onto the fluorescence microscope (Axiovert 200M; Zeiss, Vienna, Austria). After 8 min, attached mitochondria were perfused with assay buffer (20 mM HEPES, 130 mM KCl, 1 mM MgCl2, 0.5 mM KH2PO4, 10 mM succinate, 2 mM malate, 0.5 mM EGTA, 0.6 mM ATP, 0.05 mM ADP at pH 7.2) at 2 ml per min. Ca2+ uptake into single mitochondria was monitored on changing the perfusion solution from Ca2+ free buffer to a buffer containing 10 μM free Ca2+.
Free Ca2+ concentration within the lumen of the endoplasmic reticulum Ca2+ was measured with D1 (ref. 27 (link)), as described previously28 (link).
