Cytexpert software

Eyecups of PBS-perfused mice were dissected to separate the retina from adjacent tissue. Subsequently, the retinal tissue was dissociated by resuspension, and dead cells were excluded by incubating in the fixable viability dye 450 (Thermo Fisher Scientific). Cells were stained with anti-CD45, anti-CD11b, anti-F480, and CD206 (1:200; Thermo Fisher Scientific) at 4 °C for 30 min. Then, the cells were washed and analyzed using CytoFlEX (Beckman Coulter, USA) to sort retinal cells. Flow results data were obtained using the CytExpert software (Beckman Coulter, USA).
Apoptosis was examined by flow cytometry analysis. For apoptosis analysis, cells were collected, washed with PBS, and incubated with Annexin V-FITC/PI Apoptosis Detection Kit (Vazyme) for 15 min. Apoptosis was then analyzed by flow cytometry CytoFlEX (Beckman coulter, USA) according to the manufacturer’s instructions. The CytExpert software (Beckman coulter, USA) was used to analyze flow cytometry data. Results are expressed as means ± standard deviation of three independent experiments.

The experiments were performed on a CytoFLEX S Flow Cytometer (Beckman Coulter, United States) using the cytometer-interfaced CytExpert software (Beckman Coulter, Californa, United States).
The flow cytometer was set up to measure Forward Scatter signal (FSC), Violet Side Scatter signal (VSSC), Annexin-V-FITC fluorescence (FL1, exc 488 nm/em 525 nm), CD41-PE fluorescence (FL10, exc 561 nm/em 585 nm) and time. To optimize the detection of PMP, we used the SSC signal from the violet laser (405 nm, VSSC), since this signal facilitates the amplification of the differences in the refractive indices between the particles and their surrounding medium. The trigger used for PMP assessment was VSSC-Height, with a threshold in 40,000. All the signals were acquired in logarithmic amplification. Data analysis was performed using CytExpert software (Beckman Coulter) and FlowJo^™ v10.5.3 Software (BD Life Sciences).

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).