Mitochondrial Evaluation Using MitoTracker Probes

MitoTracker^® Deep Red (mitochondrial mass evaluation) or MitoTracker^® Orange CM-H2TMRos (mitochondrial membrane potential evaluation) (Life Technologies) were used to evaluate mitochondrial mass and membrane potential, respectively, as previously reported in Nigro et al. [64 (link)]. Briefly, 35 × 10⁴ cells were plated in full media for 24 h, then cells were treated or not with 2 μg/mL of C-EVs or Lep-EVs for 48 h. Next, cells were trypisinized, collected, and incubated with a MitoTracker staining solution (10 nM in PBS) for 30–60 min at 37 °C. Cells were then harvested, re-suspended in PBS and, analyzed by flow cytometry (CytoFLEX Beckman, Beckman Coulter, Milan, Italy). Data analysis was performed using CytExpert Beckman Coulter software (Beckman Coulter Milan, Italy).