Single cell suspensions were prepared from aortas as previously described.3 (link), 20 (link) Briefly, the entire aorta with surrounding perivascular fat was minced with fine scissors and digested with 1 mg/mL collagenase A, 1 mg/ml collagenase B, and 100 µg/ml DNase I in phenol-free RPMI 1640 medium with 5% FBS for 30 min at 37°C, with intermittent agitation. Fc receptors were blocked with anti-mouse CD16/CD32 for 20 min at 4°C (BD Biosciences, clone 2.4G2) prior to the staining of surface markers. The antibodies used were: PerCP-Cy5 anti-CD45; PE anti-CD34; APC anti-CD34, APC-Cy7 anti-Sca-1/Ly-6A; PE-Cy7 anti-c-kit/CD117; Amcyan anti-CD31. All aortic cells were incubated with 1.5 µl of each antibody in 100 µl of FACS buffer for 35 minutes. The cells were then washed twice with FACS buffer and immediately analyzed on a FACSCanto flow cytometer with DIVA software (Becton Dickinson). Dead cells were excluded from analysis with a fixable Violet dead cell stain. Intracellular staining was then performed with the Cytofix/Cytoperm™ Plus fixation/permeabilization solution kit (BD Biosciences) using a mouse monoclonal antibody (Abcam) conjugated to Alexa Fluor 488 or 647 (Molecular Probes). For each experiment, we performed flow minus one (FMO) controls for each fluorophore to establish gates. Data analysis was performed using FlowJo software (Tree Star, Inc.).