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Abstract
Medicine
ERRATUM NOTICE
Important: There has been an erratum issued for this article. Read more …Resident cardiac macrophages have been demonstrated to facilitate the electrical conduction in the heart. The physiologic heart rhythm is initiated by electrical impulses generated in sinoatrial node (SAN) and then conducted to ventricles via atrioventricular node (AVN). To further study the role of resident macrophages in cardiac conduction system, a proper isolation of resident macrophages from SAN and AVN is necessary, but it remains challenging. Here, we provide a protocol for the reliable microdissection of the SAN and AVN in murine hearts followed by the isolation and culture of resident macrophages.
Both, SAN which is located at the junction of the crista terminalis with the superior vena cava, and AVN which is located at the apex of the triangle of Koch, are identified and microdissected. Correct location is confirmed by histologic analysis of the tissue performed with Masson's trichrome stain and by anti-HCN4.
Microdissected tissues are then enzymatically digested to obtain single cell suspensions followed by the incubation with a specific panel of antibodies directed against cell-type specific surface markers. This allows to identify, count, or isolate different cell populations by fluorescent activated cell sorting. To differentiate cardiac resident macrophages from other immune cells in the myocardium, especially recruited monocyte-derived macrophages, a delicate devised gating strategy is needed. First, lymphoid lineage cells are detected and excluded from further analysis. Then, myeloid cells are identified with resident macrophages being determined by high expression of both CD45 and CD11b, and low expression of Ly6C. With cell sorting, isolated cardiac macrophages can then be cultivated in vitro over several days for further investigation. We, therefore, describe a protocol to isolate cardiac resident macrophages located within the cardiac conduction system. We discuss pitfalls in microdissecting and digesting SAN and AVN, and provide a gating strategy to reliably identify, count and sort cardiac macrophages by fluorescence-activated cell sorting.
Erratum
Erratum: Isolation and Culture of Resident Cardiac Macrophages from the Murine Sinoatrial and Atrioventricular NodeAn erratum was issued for: Isolation and Culture of Resident Cardiac Macrophages from the Murine Sinoatrial and Atrioventricular Node. The Authors section was updated from:
Ruibing Xia123
Simone Loy1
Stefan Kääb13
Anna Titova1
Christian Schulz123
Steffen Massberg123
Sebastian Clauss123
1University Hospital Munich, Department of Medicine I Ludwig-Maximilian-Unversity Munich (LMU)
2Walter Brendel Center of Experimental Medicine (WBex) LMU Munich
3German Center for Cardiovascular Research (DZHK), Partner Site Munich, Munich Heart Alliance (MHA)
to:
Ruibing Xia123
Simone Loy1
Stefan Kääb13
Anna Titova1
Christian Schulz123
Steffen Massberg123
Sebastian Clauss123
1University Hospital Munich, Department of Medicine I Ludwig-Maximilian-University Munich (LMU)
2Insitute of Surgical Research at the Walter Brendel Center of Experimental Medicine, University Hospital Munich, Ludwig-Maximilians-University (LMU)
3German Center for Cardiovascular Research (DZHK), Partner Site Munich, Munich Heart Alliance (MHA)
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