Isolation of Macrophage Subsets and Stromal Cells from Human and Mouse Myocardial Specimens

Summary

Presented here is a protocol to isolate various subsets of macrophages and other non-immune cells from human and mouse myocardium by preparing a single cell suspension through enzymatic digestion. Gating schemes for flow cytometry based identification and characterization of isolated macrophages are also presented.

Abstract

Macrophages represent the most heterogeneous and abundant immune cell populations in the heart and are central in driving inflammation and reparative responses after cardiac injury. How various subsets of macrophages orchestrate the immune responses after cardiac injury is an active area of research. Presented here is a simple protocol that our lab performs routinely, for the extraction of macrophages from mouse and human myocardium specimens obtained from healthy and diseased individuals. Briefly, this protocol involves enzymatic digestion of cardiac tissue to generate a single cell suspension, followed by antibody staining, and flow cytometry. This technique is suitable for functional assays performed on sorted cells as well as bulk and single cell RNA sequencing. A major advantage of this protocol is its simplicity, minimal day to day variation and wide applicability allowing investigation of macrophage heterogeneity across various mouse models and human disease entities.

Introduction

Macrophages represent the most abundant immune cell type in the heart, and they play significant roles in generating robust inflammatory and reparative responses following cardiac injury^1,2,3,4. Previously, our group identified two major subsets of macrophages in the murine heart derived from distinct developmental origins^5,6. Broadly, distinct populations of tissue resident cardiac macrophage subsets can be identified based on the cell surface expression of CCR2 (C-C motif chemokine....

Protocol

The protocol presented has been approved by the Washington University in St. Louis Institutional Review Board (#201305086). All subjects provide informed consent before sample collection and the experiments are performed in accordance with the approved study protocol. The presented protocol is performed with the approval of the Institutional Animal Care and Use Committee at Washington University School of Medicine and follows the guidelines described in the NIH Guide for the Care and Use of Laboratory Animals.

1. Preparation of Human Cardiac Tissue Specimens

1. Flush explanted hearts by cannulating the left and right coronary arter....

Results

The protocol described allows isolation of macrophages from mouse and human myocardium. Using the same protocol, but with a different staining and gating strategy, stromal cells can also be harvested from the human myocardium. FACS results presented here were acquired either on BD LSRII or BD FACS ARIA III platform. Compensation controls were generated from single color control samples from stained splenocytes. Figure 1 shows unprocessed and processed human L.......

Discussion

The protocol allows for the extraction of various macrophage subsets from human myocardium. The protocol is simple and takes 3 to 4 hours to prepare single cell suspension ready for FACS analysis. Although the protocol is relatively simple to perform, there are certain technical aspects that need to be considered which will minimize variability. Firstly, working in timely fashion with human tissue is necessary for optimal cell viability. It is important to keep the tissue in cold saline/HBSS to minimize cell death. It is.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
15 mL Conocal Tubes	Thermo Fisher	14-959-53A
40 µm Cell Strainers	Thermo Fisher	50-828-736
50 mL Conical Tubes	Thermo Fisher	352098
ACK Lysis Buffer	Gibco	A10492-01
Bovine Serum Albumin	Sigma	A2058
Collagenase 1	Sigma	C0130-1G
DAPI	Thermo Fisher	D1306
DMEM 1x	Gibco	11965-084
DNAse 1	Sigma	D4527-20KU
DRAQ5	Thermo Fisher	62251
EDTA 0.5 M pH 8	Corning	46-034-CI
Enzyme Deactivating Buffer			490 mL HBSS, 10 mL FBS, 1 g BSA
FACS Buffer			976 mL PBS, 20 mL FBS, 4 mL EDTA (0.5 M)
Fetal Bovine Serum	Gibco	A3840201
Forceps	VWR	82027-406
HBSS 1x	Gibco	14175-079
Hemostats	VWR	63042-052
Hyaluronidase type 1-s	Sigma	H3506-500MG
PBS 1x	Gibco	14190-136
Petri dishes	Thermo Fisher	172931
Razor Blade	VWR	55411-050
Scissors	VWR	82027-578