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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Medicine

Isolation and Culture of Resident Cardiac Macrophages from the Murine Sinoatrial and Atrioventricular Node

Published: May 7th, 2021

DOI:

10.3791/62236

1University Hospital Munich, Department of Medicine I, Ludwig-Maximilian-Unversity 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)

ERRATUM NOTICE

Important: There has been an erratum issued for this article. Read more …

The protocol presented here provides a step-by-step approach for the isolation of cardiac resident macrophages from the sinoatrial node (SAN) and atrioventricular node (AVN) region of mouse hearts.

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.

The sinoatrial node (SAN) physiologically initiates the electrical impulse and is, therefore, the primary pacemaker of the heart. The atrioventricular node (AVN) conducts the electrical impulse from the atria to the ventricles and also acts as a subsidiary pacemaker1. In general, generation and conduction of electrical impulses is a complex process that can be modulated by various factors2, including resident macrophage in SAN/AVN regions. A recent study by Hulsmans et al. demonstrates a specific population of cardiac resident macrophages which are enriched in the AVN and function as key players in keeping a steady heart....

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Animal care and all experimental procedures were conducted in accordance with the guidelines of the Animal Care and Ethics committee of the University of Munich and all the procedures undertaken on mice were approved by the Government of Bavaria, Munich, Germany. C57BL6/J mice were commercially obtained.

1. Preparations

  1. Prepare Cell sorting buffer (Table 1) and store at 4 °C.
    NOTE: During the whole experimental procedure, the cell sorting buffer should .......

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We describe a practical procedure for the isolation of cardiac resident macrophages specifically from the SAN and AVN region. To confirm a correct dissection, Masson's Trichrome staining and immunofluorescent HCN4-staining is performed (Figure 3)12. With this protocol, we could collect approximately 60,000 macrophages from one whole heart. Figure 4 shows the gating strategy for sorting cardiac macrophages. Live resident cardiac macrop.......

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In this manuscript, we describe a protocol for the enrichment of cardiac resident macrophages specifically from the SAN and AVN regions at high purity.

Macrophages are divided into subpopulations based on their anatomical location and functional phenotype. They can also switch from one functional phenotype to another in response to variable microenvironmental signals13. Compared to other organs such as bone marrow and liver, cardiac tissue contains a lower percentage of.......

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This work was supported by the China Scholarship Council (CSC, to R. Xia), the German Centre for Cardiovascular Research (DZHK; 81X2600255 to S. Clauss, 81Z0600206 to S. Kääb, 81Z0600204 to C.S.), the Corona Foundation (S199/10079/2019 to S. Clauss), the SFB 914 (project Z01 to S. Massberg), the ERA-NET on Cardiovascular Diseases (ERA-CVD; 01KL1910 to S. Clauss) and the Heinrich-and-Lotte-Mühlfenzl Stiftung (to S. Clauss). The funders had no role in manuscript preparation.

....

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Name Company Catalog Number Comments
Anesthesia
Isoflurane vaporizer system Hugo Sachs Elektronik 34-0458, 34-1030, 73-4911, 34-0415, 73-4910 Includes an induction chamber, a gas evacuation unit and charcoal filters
Modified Bain circuit Hugo Sachs Elektronik 73-4860 Includes an anesthesia mask for mice
Surgical Platform Kent scientific SURGI-M
In vivo instrumentation
Fine forceps Fine Science Tools 11295-51
Iris scissors Fine Science Tools 14084-08
Spring scissors Fine Science Tools 91500-09
Tissue forceps Fine Science Tools 11051-10
Tissue pins Fine Science Tools 26007-01 Could use 27G needles as a substitute
General lab instruments
Orbital shaker Sunlab D-8040
Pipette,volume 10ul, 100ul, 1000ul Eppendorf Z683884-1EA
Magnetic stirrer IKA RH basic
Microscopes
Dissection stereo- zoom microscope vwr 10836-004
Leica microscope Leica microsystems Leica DM6
Flow cytometry machine
Beckman Coulter Beckman coulter MoFlo Astrios
Software
FlowJo v10 FlowJo
General Lab Material
0.2 µm syringe filter sartorius 17597
100 mm petri dish Falcon 351029
27G needle BD Microlance 3 300635
50 ml Polypropylene conical Tube FALCON 352070
Cover slips Thermo scientific 7632160
Eppendorf Tubes Eppendorf 30121872
5ml Syringe Braun 4606108V
Chemicals
0.5 M EDTA Sigma 20-158
Acetic acid Merck 100063 Component of TEA
Agarose Biozym 850070
Bovine Serum Albumin Sigma A2153-100G
Collagenase I Worthington Biochemical LS004196
Collagenase XI Sigma C7657
DNase I Sigma D4527
Hyaluronidase Sigma H3506
HEPES buffer Sigma H4034
Bovine Serum Albumin Sigma A2153-100G
DPBS (1X) Dulbecco's Phosphate Buffered Saline Gibco 14190-094
Fetal bovine serum Sigma F2442-500ml
Penicillin − Streptomycin Sigma P4083
DMEM Gibco 41966029
Drugs
Fentanyl 0.5 mg/10 ml Braun Melsungen
Isoflurane 1 ml/ml Cp-pharma 31303
Oxygen 5L Linde 2020175 Includes a pressure regulator
Antibodies
Anti-mouse Ly6C FITC (clone HK1.4) BioLegend Cat# 128006 diluted to 1:100
Anti-mouse F4/80 PE/Cy7(clone BM8) BioLegend Cat# 123114 diluted to 1:100
Anti-mouse CD64 APC (clone X54-5/7.1) BioLegend Cat# 139306 diluted to 1:100
Anti-mouse CD11b APC/Cy7(clone M1/70) BioLegend Cat# 101226 diluted to 1:100
Anti-mouse CD45 PE (clone 30-F11) BioLegend Cat# 103106 diluted to 1:100
Hoechst 33342, Trihydrochloride, Trihydrate (DAPI) Invitrogen H3570 diluted to 1:1000
Animals
Mouse, C57BL/6 Charles River Laboratories

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Erratum

Erratum: Isolation and Culture of Resident Cardiac Macrophages from the Murine Sinoatrial and Atrioventricular Node

An 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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