Aortic Cell Suspension Preparation

To prepare aortic cell suspension, fresh descending aorta and aortic root fragments, harvested from Apoe−/− male C57BL/6 mice (18 weeks old) fed with a Western diet and normal diet, three in each group, were incubated by an enzyme mix with 0.2 mg/mL Liberase (Roche, 5,401,054,001) and 2 U/mL Elastase (Sigma-Aldrich, E1250), with HBSS as the solvent. Digestion was done by rotating at 37°C in an oven for an hour. The product was filtered through the 35 um strainer and washed with HBSS. Cells were collected by centrifugation at room temperature, 500 xg for 5 min. The supernatant was discarded and the cells resuspended with staining buffer (3% BSA and 1%NaN in PBS).
Each group of cell suspension mentioned above was divided into four parts for incubation with the following four antibodies.
Incubation was performed for 1 h at 4°C in darkness. Separate isotype controls for each antibody were also prepared. Secondary antibodies labeled with fluorescent dye were diluted with 3% BSA and used to resuspend cells at room temperature for 30 min in darkness.
Cells were washed with PBS by centrifugation at 400 g for 5 min twice. Finally, cells were resuspended with cold staining buffer (3% BSA and 1%NaN in PBS), the cell number was counted, and flow cytometry was performed. Cells were sorted by flow cytometry (CytoFLEX, Beckman Coulter) and analyzed with flow cytometer (CytoFLEX, Beckman Coulter) (version 2.0).