Mitochondrial Calcium Quantification Protocols

Levels of mitochondrial calcium were quantified by two different approaches. Cells were seeded in 96 optical well plates (Eppendorf) and incubated for 24 h or 7 d with sFLG 5 µg/mL. In the first approach, cells were loaded for 30 min with 1 µM Calcein-AM (#C1430; Thermo Fisher) and 1 mM CoCl₂ to quench the fluorescence corresponding to cytosolic Ca²⁺ as described before^{46 (link)}. After washing in fresh medium, images were acquired in a Cytation 5 Reader (Biotek) using a 20 × objective. The results were calculated with ImageJ 1.53 and are expressed as relative fluorescence units (RFUs) for each treatment (n = 3). In the second approach, cells were loaded for 30 min with 1 µM Fluo-4 and Mitotracker Red CMXRos (#M7512; Thermo Fisher). After washing in fresh medium images were acquired in a Cytation 5 Reader (Biotek) using a 20 × objective. The percentage of mitochondrial area colocalized with Fluo-4 signal was calculated with ImageJ 1.53 (colocalization plug-in). Results are expressed as the percentage of mitochondrial area colocalized per cell for each treatment (> 50 cells).