title: Preparation of Single Cell Suspensions from Mouse Aorta.
abstract: Atherosclerosis is a chronic inflammatory disease of the arterial wall characterized by lipid deposition, plaque formation, and immune cell infiltration. Innate and adaptive immune cells infiltrate the artery during development of the disease. Moreover, advanced disease leads to formation of artery tertiary lymphoid organs in the adventitia (Grabneret al., 2009; Huet al., 2015). Various and diverse types of immune cells have been identified in the aorta adventitia vs atherosclerotic plaques (Elewaet al., 2016; Galkinaet al., 2006; Lotzeret al., 2010; Mohantaet al., 2016; Mohantaet al., 2014; Mooset al., 2005; Srikakulapuet al., 2016; Zhaoet al., 2004). There are conflicting reports on the number and subtypes of immune cells in the aorta depending on the age of the animals, the protocol that is used to obtain single cell suspensions, and the dietary conditions of the mice (Campbellet al., 2012; Clementet al., 2015; Galkinaet al., 2006; Kyawet al., 2012). The number of immune cells in the aorta differs as much as tenfold using different protocols (Butcheret al., 2012; Galkinaet al., 2006; Gjurichet al., 2015; Grabneret al., 2009; Huet al., 2015). These discrepant results call for a protocol that robustly documentsbona fideaorta cells rather than those in the surrounding tissues or blood. Critical methodological hurdles include the removal of adjacent adipose tissue and small paraaortic lymph nodes lining the entire aortic tree that are not visible by the naked eye. A dissection microscope is therefore recommended. Moreover protocols of aorta preparations should ascertain that lymphocyte aggregates referred to asfat associated lymphoid clusters(FALCs) (Benezechet al., 2015; Elewaet al., 2015) that are often present at the border between the adipose tissue and the adventitia are removed before enzyme digestion. We propose - besides other approaches (Huet al., 2015; Mohantaet al., 2014) - a combination of immunohistochemical staining and fluorescence activated cell sorter (FACS) analyses from single cell suspensions to quantify the cells of interest. This protocol describes isolation of single cells from mouse aorta for FACS and other analysis.
authors.affiliation: Institute for Immunology, Ludwig-Maximilians-University Munich, Munich, Germany; Institute of Molecular Immunology, Helmholtz Zentrum Munich, Munich, Germany.
authors.forename: Desheng
authors.initials: D
authors.lastname: Hu
authors.affiliation: Institute for Cardiovascular Prevention, Ludwig-Maximilians-University Munich, Munich, Germany.
authors.forename: Changjun
authors.initials: C
authors.lastname: Yin
authors.affiliation: Institute for Cardiovascular Prevention, Ludwig-Maximilians-University Munich, Munich, Germany.
authors.forename: Sarajo K
authors.initials: SK
authors.lastname: Mohanta
authors.affiliation: Institute for Cardiovascular Prevention, Ludwig-Maximilians-University Munich, Munich, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Weber
authors.affiliation: Institute for Cardiovascular Prevention, Ludwig-Maximilians-University Munich, Munich, Germany.
authors.forename: Andreas J R
authors.initials: AJ
authors.lastname: Habenicht