Evaluating mEV Cell Entry Dynamics

To evaluate the propensity of mEVs to enter different cell types in the peripheral blood and particularly monocytes, we performed an mEV entrance experiment. Peripheral blood mononuclear cells (PBMCs) were enriched from a healthy donor as previously described^{[11 (link)]}. In an ultra-low attachment 6-well plate (Corning, Kennebunk, ME, USA), 6 × 10⁶ PBMCs were cultured in RPMI-1640 supplemented with 10% FBS at 2 × 10⁶ /mL. Ten μg of mEVs transfected with Cy-5 labelled antagomir-155 were added. Cells were incubated for 24 h and sampled at 2 h, 8 h and 24 h. Cells were immediately stained with CD8-FITC, CD4-PerCP-Cy5.5, CD14-PE, CD16-BV421 (all from BD, Becton, Dickinson and Company, Franklin Lakes, NJ, USA) for 30 min at room temperature (RT). Cells were then washed and fixed in 2% PFA for 10 min at RT, followed by washing. Flow cytometry analyses were performed on a FACSAria II flow cytometer (BD) and data were analyzed with FlowJo software (BD). At least 10,000 cells were collected for each sample. Gates were set using isotype antibodies (BD).