Megamix plus beads

CMV from H9c2 -cells or murine cardiomyocytes were diluted in PBS and counted by flow cytometry. An MV gate was defined using Megamix-Plus Beads (BioCytex, France) to include MV with a size range between 0.2 and 1 µm. 10,000 counting beads (Trucount, BD, USA) were recorded per sample and the MV concentration was calculated.
Vitality of cardiomyocytes was assessed using propidium iodide staining (PI, R&D Systems, USA) as described previously [20 (link)].

To broadly setup scatter parameters for EV and liposome detection, Megamix-Plus beads (Biocytex, Marseille, France) were acquired according to manufacturer’s instructions (threshold on FITC channel). Height (H) signals and logarithmic or bi-exponential modes were selected for all parameters.
Instrument performances were monitored by the Cytometer Setup and Tracking Module and further validated by the acquisition of Rainbow Beads (BD Biosciences, San Jose, CA, USA ) [38 (link),59 (link),60 (link)]. Data were analyzed using FACSDiva v 6.1.3 (BD Biosciences, San Jose, CA, USA), FACSuite v 1.0.6.5230 (BD Biosciences, San Jose, CA, USA) and FlowJo v 10.0.7 (TreeStar, now Becton, Dickinson and Company, Ashland, OR, USA) software.

To explore the possibility to standardize EV counts, stained samples were also acquired by using three different flow cytometers (FACSVerse, FACSCanto II, and FACSAria III; San Jose, CA, USA). All three instruments were standardized on the PE channel using Sphero Rainbow Calibration Particles (BD Biosciences, San Jose, CA, USA, cat. 559123) as previously described [38 (link)] and Megamix-Plus beads (Biocytex, Marseille, France) to broadly set scatter parameters, according to the manufacturer’s recommendations. The threshold, applied on the PE channel, was regulated until just above the background counts while measuring buffer-only samples.
To define the obtained level of standardization, MSC-derived EVs from the same samples (n = 3) were counted by using Trucount tubes (BD Biosciences, San Jose, CA, USA), using as a stopping gate the bead number (200).