Mice were deep-anesthetized at the end of experiment and their vasculature were perfused by cardiac puncture with PBS containing 20 U/ml heparin to remove blood cells from all vessels. The aortas were collected and digested as previous described 31 (link). Briefly, aortas, free of adipose tissue, were collected and weighed to control the total collected amount. The harvested aortas were minced with scissors and digested with 125 U/ml collagenase type XI, 60 U/ml hyaluronidase type I, 60 U/ml DNase1, and 450 U/ml collagenase type I in PBS containing 20 mM HEPES at 37°C for 45 minutes. Aortic cell suspensions were obtained by mashing the aorta through a 70 μm cell strainer for flow cytometry analysis. Cells were first stained with live/dead-blue dye for 30 minutes at room temperature to exclude dead cells. Then the cells were washed and co-incubated with four monoclonal antibodies: CD11b- BV421, Ly6C-APC, F4/80-PE, and CD45-APC/Cy7 (leukocyte marker) (0.25 μg/100 μl) (BD PharmingenTM, San Diego, CA) for 15 minutes in dark at 4°C. Flow cytometry analysis was performed on a LSRII analyzer (BD Biosciences). Data were analyzed using the FlowJo software (Tree Star Inc., Ashland, OR).