Dulbecco phosphate buffered saline (dpbs)

Atherosclerotic plaque tissues were washed with Dulbecco's phosphate-buffered saline (DPBS, Sigma-Aldrich, United States) within 1 hour after surgery. Each specimen was digested at 37°C for 1 hour using miscible liquids that contained collagenase type IV (GIBCO, Gaithersburg, United States), DNase (Sigma, DN25), hyaluronidase (Sigma, H3506), collagenase type XI (Sigma, C7657), and collagenase type II (Sigma, C6885). The mixture was then filtered through a 70 μm cell strainer with DPBS and washed with red blood cell (RBC) lysis buffer (BD Biosciences, United States). The dissociated cell suspension was washed once with DPBS and resuspended in 1 mL of staining buffer (DPBS containing 5% fetal bovine serum, ScienCell, United States).

Protein was isolated from hfPMVECs, as described previously (Toby et al. 2010; Cui et al. 2011; Nelin et al. 2016). Briefly, after the experiments were completed, hfPMVECs were washed with Dulbecco's Phosphate‐Buffered Saline (DPBS) (Catalog #:0303, ScienCell Research Laboratories, Inc,) and lysis solution (20 mmol/L HEPES, pH 7.4, 50 mmol/L glycerophosphate, 2 mmol/L EGTA, 1 mmol/L DTT, 10 mmol/L NaF, 1 mmol/L Na₃VO₄, 1% Triton X‐100, and 10% glycerol) was added. Thirty minutes before use, the following protease inhibitors were added to each milliliter of lysis solution: 1 μL aprotinin (10 mg/mL double‐distilled H₂O), 1 μL leupeptin (10 mg/mL double‐distilled H₂O), and 1 μL of phenylmethylsulfonyl fluoride (100 mmol/L/mL isopropanol). hfPMVECs were scraped and placed in sterile centrifuge tubes on ice. The samples were centrifuged at 20,000g for 15 min at 4°C. The supernatant was stored at −80°C for subsequent western blot analysis. Total protein concentration was determined by the Bradford method using a commercially available assay (Bio‐Rad, Hercules, CA) as described previously (Toby et al. 2010; White et al. 2017).

Human spleen endothelial cells (hSEC) were purchased from ScienCell (Cat. #5500). hSEC were cultured in fibronectin-coated culture vessel using Endothelial Cell Medium (ScienCell) at 37 °C, 5% CO₂. Cells were dissociated with 5 times diluted 0.05% trypsin-EDTA solution (Sigma) in DPBS (ScienCell), and seed in new flask up to 15 times. For NF-kB translocation assay, 2.5 x 10⁴ hSEC per well were seeded in 500 μL of EV-depleted Endothelial Cell Medium on coverslips (Nunc™) in 24-well plates (Merck Millipore), and incubated with PvEVs, hEVs (20 μg/mL), or medium alone (Ctr) at 37 °C, 5% CO₂ for 30 min. For qRT-PCR, the cells seeded in 24-well plates (Merck Millipore) were incubated with PvEVs, hEVs (20 μg/mL), or medium alone (Ctr) at 37 °C, 5% CO₂ for 24 h. Each condition was performed in triplicate and qRT-PCR was done in technical triplicates.