F 7000 spectrophotometer

For free [Ca²⁺]_i measurements, cells were loaded with ratiometric Ca²⁺ indicator Fura-2 (F1201, Thermo Fisher Scientific). Cells were twice washed with PBS and resuspended in a loading solution (HBSS, 0.01% pluronic acid, 2 µM Fura-2/AM), incubated for 30 min at room temperature protected from light, washed, and resuspended in HBSS. Changes in fluorescence were recorded with a F7000 spectrophotometer (Hitachi High-Technologies). Measurements were realized in quartz cuvettes, containing 1.5 × 10⁶ cells/mL. Loaded cells were excited alternately at 340/380 nm and the fluorescence emission was collected every 2 s at 510 nm. Fluorescence was recorded by means of the FL-solutions software. Maximum and minimum free [Ca²⁺] levels were determined at the end of each experiment by adding 0.3% Triton X-100 and consequent addition of EGTA to a final concentration of 35 mM, respectively. Free [Ca²⁺]_i was calculated by using the following equation: ${\left[{\mathrm{Ca}}^{2+}\right]}_{\mathrm{i}}=K_{\mathrm{d}}\left(R-R_{\mathrm{MIN}}\right)∕\left(R_{\mathrm{MAX}}-R\right)$ where R stays for the ratio of fluorescence intensity upon excitation at 340 to that at 380 nm and R_MIN and R_MAX correspond to maximal and minimal values of this ratio, determined as described above^{43 (link)}.
In some experiments Ca²⁺-free HBSS was used (NaCl 143 mM, KCl 6 mM, MgSO₄ 5 mM, HEPES 20 mM, BSA 0.1%, glucose 5 mM, EGTA 1 mM, pH 7.4, ≈300 mOsm).

Freshly isolated mitochondrial samples (50 μg of protein per sample) were incubated with Rhod2 (2 μM) over 30 min, washed by centrifugation (12500 × g, 5 min) and resuspended in experimental buffer (KCl 125 mM, KH₂PO₄ 1 mM, TrisHCl 10 mM, glutamate 5 mM, malate 2.5 mM, EGTA 1 mM, CaCl₂ 0.7 mM). Samples were placed in a quartz cuvette, and fluorescence was evaluated by F7000 spectrophotometer (Hitachi High-Technologies), using excitation at 552 nm and collecting the fluorescence at 581 nm. Data was recorded using the FL-Solution software (Hitachi). [Ca²⁺]_m change was evaluated by taking fluorescence for each acquisition in relation to the initial fluorescence value, F/F0.

Data analysis was based on the fact that the maximum fluorescence excitation of the mtKeima protein occurs at 586 nm at acidic pH and 440 nm at neutral pH, while the emission spectrum is independent of pH, with a maximum at 620 nm [23 (link)]. Accordingly, images were acquired using a confocal microscope (LSM 700, Carl Zeiss, Jena, Germany) equipped with 405 and 555 nm lasers and 40×/63×/100× oil-immersion objectives. Images were generated with Zen lite 3.0 software (Carl Zeiss, Jena, Germany). Raw images were further processed using ImageJ 1.53t software (NIH). For quantitative analysis, transfected cells were analyzed through spectrofluorometry (F7000 spectrophotometer, Hitachi High-Technologies, Tokyo, Japan). The samples were excited in the 300–700 nm range, and the fluorescence intensity values at 620 nm were estimated. The ratio, obtained at 620 nm for 586 nm and 440 nm excitation (586/440), reflected the mitophagy progression. Data were normalized to control (untreated) samples and averaged, and the data from at least four independent experiments were graphed using a GraphPad prism.