Anti cd45 antibody and fixable live dead cell stain

Cellular composition of aortas was determined in the Cell Phenotyping Core of the UCSD PPG on Role of Immune Mechanisms in Inflammation and Atherosclerosis, under the direction of K. Ley, using established techniques³⁷ . In brief, 6 aortas from 16-week chow-fed Ldlr^−/−, 6 from HFD-fed Ldlr^−/− and 5 from Ldlr^−/−/E06-scFv mice were dissected following heparin PBS perfusion and adventitia carefully removed. The intact aortas were incubated for one hour with an Aorta Dissociation Enzyme stock solution and single cell suspensions prepared from the digested aorta by shearing the aortas apart and passing cells through a 70μm cell strainer into 5ml polypropylene FACS tubes (BD Falcon). The cells were pelleted by centrifugation (400×g, 5 minutes, 4°C), resuspended in 1ml of FACS buffer (PBS supplemented with 1% BSA and 0.05% NaN₃), counted and assessed for viability using trypan blue in a hemocytometer. Cells were stained on ice for 30 min with the panel of antibodies below, washed twice with FACS buffer and then analyzed at La Jolla Institute for Allergy and Immunology using a FACSAria analyzer. Anti-CD45 antibody and fixable live-dead cell stain (Invitrogen, Molecular Probes) was added to all samples to allow for gating of live CD45+ leukocytes and cells were sorted with the panel of antibodies listed in Table in Supplemental Information.