Forty-eight hours later, CM was collected and extracellular vesicles (EV) were isolated, essentially as described.29 Briefly, cells and cell debris were removed by serial centrifugation of CM and decantation of EV-containing supernatant: 300 for 10?min, 2,000 for 20?min, and 10,000??g for 30?min (all at 4C). Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS), 1% Pen-Strep, and 400?g/mL G418 (Thermo Rabbit Polyclonal to ZNF446 Fisher Scientific, Waltham, MA). Bovine aortic EC (BAEC) (Cell Applications, San Diego, CA) and bovine aortic SMC (BASMC) (Cell Applications) were maintained in high-glucose DMEM (GIBCO, Grand Island, NY), 10% FBS (VWR Life Science Seradigm, Radnor, PA), and 1% Pen-Strep (GIBCO) and they were used between passages 3 and 7. Human THP-1 monocytes (ATCC, Manassas, VA) were maintained in high-glucose RPMI-1640 (ATCC) containing 10% FBS and 1% Pen-Strep and were differentiated into macrophages by treatment with phorbol myristate acetate (100?ng/mL) for 72?h. All cells were Raphin1 acetate cultured at 37C with 5% CO2. We used BAEC because they are easily propagated, which is necessary to produce the large amount of exosome-containing medium that we anticipated would be needed for these experiments. We used BASMC, because they are a useful experimental model of cellular cholesterol loading and inducible cholesterol efflux.27,28 We used THP-1 cells because they are also a useful model of cholesterol loading and inducible cholesterol efflux, and they express both miR-33a-5p and ABCA1 (useful as positive controls).22 For transduction, BAEC were grown to 80C90% confluency; then, they were exposed to viral vectors (1??1010 viral particles (vp)/mL in growth medium) for 6?h. The vector-containing medium was then removed; cells were washed with phosphate-buffered saline (PBS) and fed with either growth medium or serum-free DMEM. This BAEC-conditioned medium (CM) was collected 48?h later and either used immediately or stored at ?80C. Isolation of extracellular vesicles from CM of untransduced BAEC BAEC were grown to 80C90% confluency in 150-mm dishes, washed with PBS, and fed with serum-free DMEM (8?mL per 150-mm dish). Forty-eight hours later, CM was collected and extracellular vesicles (EV) were isolated, essentially as described.29 Raphin1 acetate Briefly, cells and cell debris were removed by serial centrifugation of CM and decantation of EV-containing supernatant: 300 for 10?min, 2,000 for 20?min, and 10,000??g for 30?min (all at 4C). EV were then pelleted from the CM by ultracentrifugation (100,000 for 90?min at 4C). The post-ultracentrifugation CM supernatant (termed EV-depleted CM) was either discarded or stored at ?80C for future use, as a control. The pelleted EV were gently resuspended in PBS and re-pelleted by ultracentrifugation, as described earlier. The post-ultracentrifugation PBS supernatant was then either discarded or stored at ?80C for future use, as a control. The pelleted EV were either used immediately (for nanoparticle tracking analysis, imaging, or RNA/protein analyses) or gently resuspended in 50?L PBS and stored at ?80C. EV size and appearance Freshly isolated EV, EV-depleted CM, and post-ultracentrifugation PBS supernatant were evaluated with nanoparticle tracking analysis (NanoSight NS300 instrument; Malvern Instruments, Malvern, UK), which measures particle concentration and diameter.30 Briefly, pelleted EV samples were vortexed and serially diluted at a ratio of 1 1:6,000C1:15,000 with molecular-grade double-distilled water. This diluent also served as a negative control. Three freshly isolated EV samples were measured with nanoparticle tracking, and mean values were calculated. We used an established protocol to image EV by transmission electron microscopy.29 Briefly, the remainder of the three EV samples used for nanoparticle tracking analysis were frozen, then thawed, and pooled (100?L total volume). We added an equal volume of 4% paraformaldehyde; then, we deposited the mixture by airfuge on Formvar-carbon coated transmission electron microscopy grids (Ted Pella, Redding, CA). Samples were embedded and contrasted by treatment with uranyl-oxalate solution (pH 7; Electron Microscopy Sciences, Hatfield, PA) for 5?min, followed by treatment with methyl-cellulose-uranyl-acetate (Sigma-Aldrich, St. Louis, MO) on ice for 10?min. Transmission electron micrographs were acquired by using a JEM-1400 transmission electron microscope (JEOL, Tokyo, Japan) at 120kV along with an Ultrascan 1000XP digital camera (Gatan, Pleasanton, CA). Molecular cloning and HDAd generation, amplification, and characterization We purchased two plasmids (MZIP33a-PA-1 and XMIRXP-NT; SBI, Palo Alto, CA). MZIP33a-PA-1 contains an expression cassette in which the H1 promoter31 drives transcription of an antagomiR targeted at miR-33a-5p.22 XMIRXP-NT contains Raphin1 acetate an expression cassette in which the H1 promoter Raphin1 acetate drives transcription of a non-targeted scrambled control shRNA.22 XMIRXP-NT also includes an X-motif DNA sequence that, when transcribed, increases uploading of shRNA transcripts into exosomes.32 Both expression cassettes also contain a 5T (TTTTT) termination signal.33 We used these plasmids as templates for PCR (Q5? High-Fidelity DNA Polymerase; New England Biolabs, Ipswich, MA; see Supplementary Table S1 for primer sequences) that generated three expression cassettes.