C57BL/6, ApoE−/−or ApoE−/−(WD) mice were anesthetized and their vasculature was perfused by cardiac puncture with PBS containing 20 U/ml of heparin to remove blood from all vessels. Under a dissection microscope, we carefully harvested whole aortas by pulling off all of the adipose tissue and collecting all aortic layers including the adventitia. To fully characterize the collected aortas, we measured aortic wall thickness and adventitial thickness in paraffin-cut sections. In addition, the dissected aortas were weighed to control the total amount of collected aortic tissues. Harvested aortas were microdissected and digested with 125 U/ml collagenase type XI, 60 U/ml hyaluronidase type I-s, 60 U/ml DNase1, and 450 U/ml collagenase type I (all enzymes were obtained from Sigma-Aldrich) in PBS containing 20 mM Hepes at 37°C for 1 h. A cell suspension was obtained by mashing the aorta through a 70-μm strainer. Cells were incubated with Abs for 20 min at 4°C, washed twice, and incubated with secondary Abs for an additional 20 min. After washing, immunofluorescence was detected by flow cytometry (FACSCalibur or CyanADP), data were analyzed using WinMDI (The Scripps Research Institute) or FlowJO (Tree Star Inc.) software. Abs used were as follows: allophycocyanin (APC)-Cy7 or PE-Texas red-CD45, FITC, PE or PerCP-CD3, FITC, APC-TCRβ, PE-Cy5 or APC-CD19, PE-B220, PE-I-Ab, PerCP-Cy5.5-Mac-1, PE or APC-CD11c (all Abs were obtained from BD Biosciences) and FITC-CD68 (Serotec). In some experiments, the aortas from two to three mice were pooled and analyzed. In some experiments, LN or spleens from C57BL/6 mice were collected and split in two parts: one part of pLN (or spleen) was treated with the enzyme cocktail (see previous paragraph) and the other was kept at 4°C. After 1 h, the expression of CD45, TCR, CD19, I-Ab antigens was determined by flow cytometry.