Percy5.5 cd11b

Single-cell suspensions were prepared from the mouse aortas as described^{20 (link)}. Briefly, mice were anesthetized and perfused through the heart with PBS containing 10 U ml⁻¹ heparin to remove blood from all vessels. After careful removal of the perivascular adipose tissue, the aortas were minced into small pieces and digested with 125 U ml⁻¹ collagenase type XI, 60 U ml⁻¹ hyaluronidase type I-s, 60 U ml⁻¹ DNase1, and 450 U ml⁻¹ collagenase type I (all enzymes were obtained from Sigma-Aldrich) in PBS containing 20 mM Hepes at 37°C for 3 hrs. For separating adventitia and media, the aortas were predigested in enzyme cocktail (300U ml⁻¹ Collagenase I and 10U ml⁻¹ Elastase in PBS) at 37°C for 15 minutes and then the adventitia was carefully peeled away from the aorta. After filtering through a 70 μm filter, cells were resuspended in FACS buffer, and incubated with Fc-blocking antibody (eBioscience) for 15 min on ice before being stained with specific antibodies. Antibodies used were as follows: FITC-CD45.2, PE-CD11c, APC-CD3, APC-Cy7-CD19, PE-Cy7-NK1.1, PerCy5.5-CD11b (all antibodies were obtained from Biolegend and used at 1:100 dilution). Cells were also stained with propidium iodide. After washing, immunofluorescence was detected by an LSR II (BD Biosciences). Data were analyzed using FlowJo (Tree Star Inc.) software.