Flow Cytometry Analysis of Vascular Leukocytes

Flow cytometry for single-cell suspension of femoral arteries was performed to analyze adherent or infiltrated leukocytes in vessel. Wt mice fed NC or HFD for 4 weeks and CCR2^−/− mice fed HFD for 4 weeks were perfused from the left ventricle with PBS (−). Femoral arteries were obtained carefully and vessels from two mice were pooled. The collected vessels were digested by 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 with shaking40 (link). The single cell suspensions were filtered with a 36 μm-nylon mesh and centrifuged at 2 g for 5 min at 4 °C. The pellets were reacted with rat anti-mouse CD11b-FITC (Biolegend, Inc.), rat anti-mouse CD11c-PE(Biolegend, Inc.), rat anti-mouse CD45-PerCP (Biolegend, Inc.), and anti-mouse/rat CCR2-APC (R&D Systems Inc.) at 4 °C for 20 min. For washing, the cell suspensions were added to 2 mM EDTA in PBS (−) and centrifuged at 2 g for 5 min at 4 °C. After fixation of the cells with 1% paraformaldehyde in PBS (−), flow cytometry analysis was performed.