M1000 fluorimeter

We used JC-1 dye (T3168, Thermo Fisher Scientific) in mitochondria isolated from 24 hpf zebrafish. JC-1 is a cationic dye that exhibits potential-dependent accumulation in mitochondria, indicated by a fluorescence emission shift from green (∼525 nm) to red (∼590 nm). We presented results as a ratio of spectra. Isolated mitochondria from 24 hpf zebrafish embryos were transferred to a 96-well non-transparent plate with a flat bottom (Greiner Bio-One), with 80 μl per well (16 μg). Measurement of the fluorescence intensity was performed in the Tecan M1000 fluorimeter (Tecan Trading AG) with excitation 490 nm and emission in two channels: 530 and 590 nm at 20°C. Experiments were conducted until fluorescence showed plateau lines.

We compared Ca²⁺ uptake capacity in mitochondria purified from 24 hpf wt, pink1^−/−, mcu^−/− and (pink1; mcu)^−/− zebrafish. The ability of the assay to detect mitochondrial calcium influx was evaluated using treatment with mitochondrial calcium influx blocker Ruthenium Red and CCCP, which causes mitochondrial calcium efflux due to its mitochondrial uncoupling property. Extramitochondrial-free Ca²⁺ was monitored in the presence of purified mitochondria using Oregon Green 5N hexapotassium salt and the measurement of the fluorescence intensity was performed in the Tecan M1000 fluorimeter (Tecan Trading AG) with parameters: ex/em, 490 nm/525 nm; bandwidth, 15 nm; 20°C. After approximately 20 s of measurement, CaCl₂ (final concentration=10 µM) was added into wells, supplemented with different reagents based on the variant [0.2 μM CCCP (C2759, Sigma-Aldrich); 10 μM Ruthenium Red (00541, Sigma-Aldrich)]. After addition of reagents, measurements were carried out until fluorescence intensity reached the plateau. Results were plotted onto the graph as an absolute fluorescence level.