Analyzing Ex Vivo Metabolic Flux in Splenic and Cardiac Macrophages and Bone Marrow Monocytes

Summary

Here, we describe in detail methods for extracting macrophages from the bone marrow, spleen, and infarcted heart, and subsequently assessing metabolic flux in live cells.

Abstract

Metabolic reprogramming is a hallmark of monocyte/macrophage activation and polarization between pro- and anti-inflammatory states. For example, pro-inflammatory (i.e., M1-like) monocytes/macrophages display more reliance on anaerobic glycolysis and less reliance on mitochondrial oxidative phosphorylation, whereas anti-inflammatory (M2-like) macrophages display more reliance on glucose and fatty acid oxidation in the mitochondria. Here, we describe in-depth protocols for extracting macrophages from the two major monocyte/macrophage reservoirs in the body, the spleen and bone marrow, as well as injured tissues such as the heart following myocardial infarction.

Macrophages or monocytes are extracted by immunomagnetic sorting by using antibody-tagged microbeads, which easily bind to cells without compromising their phenotypes. The extracted cells are then cultured in 96-well plates, followed by extracellular flux analysis using a metabolic flux analyzer. Both glycolysis and mitochondrial oxidative phosphorylation can be measured simultaneously in small numbers of cells (as little as 2-3 × 10⁵ cells). This method can easily be performed in 1 day and produces reliable and repeatable results. Ultimately, these methods help to enhance our understanding of metabolic changes during immune and inflammatory responses to injury and disease, which could lead to the development of novel therapeutic targets for immunometabolic pathways.

Introduction

Immunometabolism is a blossoming field that studies the role of metabolic reprogramming in immune cells across different pathological disease and injury states. Macrophages are a key part of the innate immune system that play critical roles in inflammation, response to infection, antigen presentation, and wound healing¹. Understanding how macrophages polarize between pro- and anti-inflammatory (M1-like and M2-like) subsets across different disease states is an area of ongoing and intense investigation. Recent studies have identified metabolic reprogramming as a key mechanism underlying macrophage polarization. The current paradigm is that, broa....

Protocol

The methods below describe protocols for extracting macrophages and performing ex vivo analysis of metabolic flux from the infarcted heart following MI, the spleen, and the bone marrow (Figure 1). For the MI heart and spleen, the extraction method used is identical. All protocols involving mice were approved by the University of Mississippi Medical Center Institutional Animal Care and Use Committee (Protocol #1371, Mouton).

1. Extraction of macrophages from the infarcted heart and spleen

NOTE: Extraction of tissue macrophages uses a negative selection strategy....

Results

Typical cell numbers obtained from the different tissues depend on the size, age, and sex of the animal. For an adult male mouse (i.e., 16 weeks old, ~30 g), the spleen may yield 3.0-4.0 × 10⁶ macrophages, while the bone marrow (two tibias and two femurs) typically yields 1.0-1.5 × 10⁶ monocytes (Figure 2A). The infarcted heart typically yields high numbers as well, depending on the day post MI^4,5,

Discussion

Our method details the rapid extraction of macrophages from bone marrow, spleen, and the infarcted heart, which can then be used to perform downstream analyses such as extracellular metabolic flux analysis. The combination of these two methods is a powerful tool that can quantify metabolic changes in macrophages under different disease or injury states, or metabolic states such as exercise. While our method focused on bone marrow and splenic macrophages, other macrophage reservoirs can be used, such as the peritoneal com.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Anti-CD11b Microbeads UltraPure, mouse	Miltenyi Biotec	130-126-725
Anti-Ly6G Microbeads UltraPure, mouse	Miltenyi Biotec	130-120-337
Collagenase type II	Worthington	NC9522060
Dnase type I	MillPore Sigma	11284932001
GentleMACS Octo Dissociator	Miltenyi Biotec	130-096-427
GentleMACS C Tubes	Miltenyi Biotec	130-093-237	MACS cell dissociation tubes
Hank balanced salt solution	Fisher Scientific	14-025-076
LS Magnetic Columns	Miltenyi Biotec	130-042-401
MACS MultiStand	Miltenyi Biotec	130-042-303
MS Magnetic Columns	Miltenyi Biotec	130-042-201
Monocyte Isolation Kit Bone Marrow, mouse	Miltenyi Biotec	130-100-629	includes 1 mL of monocyte biotin-antibody cocktail, 2 mL of anti-biotin microbeads, 1 mL of FcR blocking reagent
OctoMACS Separator	Miltenyi Biotec	130-042-108
Pre-separation filters (30 µm)	Miltenyi Biotec	130-041-407
QuadroMACS Separator	Miltenyi Biotec	130-091-051
Red Blood Cell Lysis Solution (10x)	Miltenyi Biotec	130-094-183
Seahorse XFe96 Analyzer	Agilent
Seahorse XFe96 FluxPak	Agilent	103792-100	18 Pro sensor cartridges, 18 Pro Cell Cutlure Microplates, 1 bottle of Seahorse XF Calibrant Solution
Seahorse XF Glycolysis Stress Test Kit	Agilent	103020-100	6 single-use pouches, each with glucose, oligomycin, and 2-deoxy-D-glucose
Seahorse XF Mitochondrial Stress Test Kit	Agilent	103015-100	6 single-use pouches, each with oligomycin, FCCP, and rotenone/antimycin A
Seahorse XF RPMI Medium, pH 7.4, 500 mL	Agilent	103576-100	no phenol red, bicarbonate, glucose, pyruvate, or glutamine