Flow Cytometric Analysis of Aortic and Blood Cells

Flow cytometry of aortas and whole blood was performed as described previously [33 (link), 68 (link)]. Briefly, cleaned aortic vessels were digested with liberase™ (1 mg/mL, Roche, Basel, Switzerland) for 30 min at 37 °C and passed through a cell strainer (70 μm) to yield a single-cell suspension. Single-cell suspensions were treated with Fc-block (anti-CD16/CD32), washed, and surface stained with CD45 APC-efluor 780 (#47-0451-82, 2 µg/mL), NK1.1 PE-Cy7 (#25-5941-81, 2 µg/mL), F4/80 APC (#17-4801-82, 4 µg/mL) from eBioscience (San Diego, CA) and TCR-β V450 (#560706, 2 µg/mL), CD11b PE (#553311, 2 µg/mL), Ly6G FITC (#551460, 5 µg/mL), and Ly6C PerCP-Cy.5.5 (#560525, 2 µg/mL) from BD Biosciences (San Diego, CA). Dead cells were excluded by staining with Fixable Viability Dye eFluor506 (#65–0866-14, 1:1000, eBioscience, San Diego, CA). Based on a live gate, events were acquired and analyzed using a BD FACS CANTO II flow cytometer (Becton Dickinson, Franklin Lakes, NJ) and FACSDiva software (Becton Dickinson, Franklin Lakes, NJ), respectively. Gating was performed according to a previously published strategy [63 (link)].