Abstract 478: Single-Cell Isolation and Analysis of Viable Proliferating Macrophages From Atherosclerotic Plaques
INTRODUCTION: We have recently shown that the proliferation of macrophages within the atherosclerotic plaque, rather than the recruitment of new monocytes from the blood is the key driver of plaque growth in established atherosclerosis. The study of proliferating macrophages has been hampered by the lack of a technique for identifying proliferating macrophages and isolating them from the atherosclerotic plaque in a viable state.
OBJECTIVE: Develop and validate a process for identifying proliferating macrophages in atherosclerotic lesions, and isolating them as single, viable cells, then perform a molecular characterization of these cells.
METHODS: ApoE-/- and LDLR-/- mice were fed a diet high in fat and cholesterol. Atherosclerotic aortas were collected, and cells were isolated by mechanical and enzymatic dissociation. Cells were stained using fluorescent markers and DNA-binding dyes, then analyzed and isolated by fluorescence-activated cell sorting.
RESULTS: Mincing and enzymatic digestion in collagenase I, collagenase XI, DNAse, and hyaluronidase optimally isolates macrophages from aortic explants. Cell-surface staining with fluorescent antibodies against B220, F4/80, Ly6c, MHC II, and CD11b allows identification of lesional macrophages. DNA staining with Vybrant DyeCycle Violet allows identification of proliferating cells. Combined, these processes allow for the FACS-based sorting of viable proliferating macrophages from atherosclerotic plaque, and subsequent RNA or protein analysis, or culture.
CONCLUSIONS: The process described here allows the first-ever viable isolation of proliferating macrophages from atherosclerotic plaques. This will permit the investigation of molecular targets to interfere with the pathogenesis of atherosclerosis.
Author Disclosures: C.C.J. Zavitz: None. S. Ensan: None. C.S. Robbins: None.
- © 2014 by American Heart Association, Inc.