Isolation and Characterization of Endothelial Extracellular Vesicles

Rat LMVECs were transiently transfected, using Polyjet reagent, with plasmid cDNA encoding GFP-tagged constructs: wild-type (p18^wt) and non-endosomal binding (p18^{N39 (link)}) p18, dominant active (Rab4^Q67L), and non-endosomal binding (Rab4^{S22 (link)N}) Rab4, or GFP vector control. Alternatively LMVECs were transiently transfected with p18 or scrambled control siRNA (300 nM) using DharmaFect4. Overexpression of constructs was assessed by GFP fluorescence quantified using the Victor fluorometer (Perkin Elmer). At 42 h post-transfection, cells were exposed to LPS (1 µg/ml) for a further six hours. Media from cells was then removed and centrifuged at 300 × g, 4℃ for 10 min twice, with pellet discarded each time. Supernatant was then centrifuged at 100,000 × g, 4℃ for one hour. The pellet, comprising of EDEVs, was resuspended in sterile Dulbecco's PBS (DPBS) for permeability assays and quantification using qNano Gold (Izon Science) at 0.5 V, 45 mm stretch, 15 mbar pressure, using the NP200 pore and normalized to media as a control, RNAlater for miRNA analysis, or lysed for protein concentration analysis.^{23 (link),24 (link)}