Replication Data for: mitochondrial potential of "Replicative Senescence in Mesenchymal Stem Cells: An In Vitro Study on Mitochondrial Dynamics and Metabolic Alterations"

Changes in mitochondrial membrane potential were analyzed by staining bMSCs with 10 µg/mL JC-1 (T3168, Invitrogen, Thermo Fisher Scientific). This probe evaluates mitochondrial potential by measuring fluorescence intensities of red-shifted aggregates (in functional mitochondria) and green-shifted JC-1 (in damaged mitochondria) monomers. Cells seeded on a 96-well black plate (Costar, Sigma-Aldrich) (6 × 103 cells/well) were incubated for 15 min at 37 °C in the dark with JC-1 (λex/λem red = 535/590 nm; λex/λem green = 485/530 nm). Fluorescence was acquired at Varioskan LUX Multimode Microplate Reader (Thermo Fisher Scientific). The red/green ratio was calculated for each sample. Mitochondrial membrane depolarization was assessed by changes in the JC-1 red/green fluorescence ratio, with a decreased ratio indicating a reduction in mitochondrial membrane potential [22]. Cells cultured on microscope slides (631-0149, VWR International S.r.l.) and incubated with JC-1 were also imaged using a 40× oil-immersion objective on an Leica SP8 confocal microscope (Leica Microsystems).