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Summary

Abstract

Introduction

Protocol

Representative Results

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Acknowledgements

Materials

References

Immunology and Infection

Isolation and Identification of Extravascular Immune Cells of the Heart

Published: August 23rd, 2018

DOI:

10.3791/58114

1Toronto General Hospital Research Institute, University Health Network (UHN), 2Dept of Laboratory Medicine and Pathobiology, University of Toronto, 3Dept of Immunology, University of Toronto, 4Peter Munk Cardiac Centre, 5Ted Rogers Centre for Heart Research

This protocol presents a simple and efficient method to isolate, identify and quantify immune cells residing in the myocardium of mice during steady state or inflammation. The protocol combines enzymatic and mechanical digestion for the generation of a single cell suspension that can be further analyzed by flow cytometry.

The immune system is an essential component of a healthy heart. The myocardium is home to a rich population of different immune cell subsets with functional compartmentalization both during steady state and during different forms of inflammation. Until recently, the study of immune cells in the heart required the use of microscopy or poorly developed digestion protocols, which provided enough sensitivity during severe inflammation but were unable to confidently identify small — but key — populations of cells during steady state. Here, we discuss a simple method combining enzymatic (collagenase, hyaluronidase and DNAse) and mechanical digestion of murine hearts preceded by intravascular administration of fluorescently-labelled antibodies to differentiate small but unavoidable intravascular cell contaminants. This method generates a suspension of isolated viable cells that can be analyzed by flow cytometry for identification, phenotyping and quantification, or further purified with fluorescence-activated cell sorting or magnetic bead separation for transcriptional analysis or in vitro studies. We include an example of a step-by-step flow cytometric analysis to differentiate the key macrophage and dendritic cell populations of the heart. For a medium sized experiment (10 hearts) the completion of the procedure requires 2–3 h.

Different forms of myocardial stress or injury, including ischemic (ischemia reperfusion or myocardial infarction) and non-ischemic (hypertension or myocarditis), promote the recruitment of inflammatory cells with reparative and protective, but also pathogenic properties. As early as 1891, Romberg first described the presence of cellular infiltrates in the myocardium of patients infected with typhus and scarlet fever1. However, the detailed study of cardiac immune cells required the development of more advanced immunophenotyping techniques. As a consequence, only recently have we started to understand that a diverse population of immune cells w....

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Ethics statement: This protocol has been reviewed and approved by the Animal Care Committee at the University Health Network (Toronto, Canada) and is in compliance with the Canadian Council on Animal Care.

1. Buffer Preparation

  1. Prepare HBB buffer (Hank's Balanced Salt Solution (HBSS), 2% heat inactivated bovine serum, 0.2% bovine serum albumin). Add 10 mL of heat inactivated bovine serum and 1 g of bovine serum albumin to 500 mL of HBSS. Filter sterilize through a 0.2 µm f.......

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To date, no good method has been designed to isolate immune cells from other cardiac cell components, such as cardiomyocytes. Hence, analysis of the cardiac single cell suspension by flow cytometry requires a pre-gating with CD45 to identify the immune cell populations, followed by single cell and small size exclusion gating (Figure 1A). Alternatively, a viability staining can be performed to exclude dead cells. Small size exclusion and viabi.......

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Myocardial inflammation, or myocarditis, is a feature of most cardiovascular diseases. However, the myocardium is not devoid of its own immune components in non-disease states. During steady state, many immune cells reside in the myocardium and play the essential roles of maintenance and protection. The characterization of these diverse populations of cells would not have been possible without methods such as the one presented in this protocol.

A combination of mechanical and enzymatic digesti.......

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This work was supported by the Canadian Institutes of Health Research (148808 and 148792), SE was supported by a Heart and Stroke Foundation, personnel award from the Ontario Provincial Office, March of Dimes, Ted Rogers Centre for Heart Research and the Peter Munk Cardiac Centre. XCC holds a CIHR Banting Fellowship. LA holds a Heart & Stroke/Richard Lewar Studentship Award.

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Name Company Catalog Number Comments
Phosphate buffered saline Wisent 311-010-CL 1x PBS
21Gx 1 1/2 (0.8mmx40mm) PrecisionGlide Needle BD 305167
Hank’s Balanced Salt Solution Wisent 311-511-CL 1x HBSS
Bovine serum albumin Sigma-Aldrich A4503-50G
Bovine serum Sigma B9433
0.5M Ethylenediaminetetraacetic acid BioShop EDT111
Vacuum filter , 0.2 µm Filtropur V50 Sarstedt 83.1823.001
28G 1/2 1 cc insulin syringe BD 329424
60 mL syringe BD 309653
Dulbecco's Modified Eagle Medium Wisent 319-005-CL 1x DMEM with 4.5 g/L glucose and L-Glutamine and Sodium Pyruvate
Collagenase I Sigma C0130 from Clostridium histolyticum
Hyaluronidase type I-S Sigma H3506
DNase-I Sigma D4513 from bovine pancreas
Cell strainer, 40 µm Nylon Falcon 352340
Ammonium-Chloride- Potassium (ACK) lysis buffer Lonza 10-546E
Alexa Fluor 700 anti-mouse/human CD11b Biolegend 101222 1:250 dilution
APC/Cy7 anti-mouse Ly-6c Biolegend 128025 1:250 dilution
APC anti-mouse CD103 Biolegend 121414 1:250 dilution
Brilliant Violet 605 anti-mouse CD11c Biolegend 117334 1:250 dilution
PE anti-mouse Ly-6G Biolegend 127607 1:250 dilution
Pacific Blue anti-mouse I-Ab Biolegend 116422 1:250 dilution
FITC anti-mouse CD64 (FcgRI) Biolegend 139315 1:250 dilution
PE/Cy7 anti-mouse CD45 Biolegend 103113 1:250 dilution
PerCP/Cy5.5 anti-mouse CD45 Biolegend 103132 1:40 dilution
TruStain fcX (anti-mouse CD16/32) Biolegend 101320 1:100 dilution
True-Stain Monocyte Blocker Biolegend 426101 1:20 dilution
FlowJo V10 TreeStar Inc https://www.flowjo.com/solutions/flowjo
Mouse: Batf3-/- The Jackson Laboratory JAX: 013755

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