Bone Marrow-derived Macrophage Production

Summary

Macrophages have long been recognized as a critical component of the innate and adaptive immune responses. The recent explosion of knowledge pertaining to evolutionary, genetic, and biochemical aspects of the interaction between macrophages and microbes has renewed scientific attention to macrophages. This article describes a method to differentiate macrophages from mouse bone marrow.

Abstract

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because of their powerful microbicidal activities. Macrophages are widely distributed throughout the body and are present in the lymphoid organs, liver, lungs, gastrointestinal tract, central nervous system, bone, and skin. Because of their repartition, they participate in a wide range of physiological and pathological processes. Macrophages are highly versatile cells that are able to recognize microenvironmental alterations and to maintain tissue homeostasis. Numerous pathogens have evolved mechanisms to use macrophages as Trojan horses to survive, replicate in, and infect both humans and animals and to propagate throughout the body. The recent explosion of interest in evolutionary, genetic, and biochemical aspects of host-pathogen interactions has renewed scientific attention regarding macrophages. Here, we describe a procedure to isolate and cultivate macrophages from murine bone marrow that will provide large numbers of macrophages for studying host-pathogen interactions as well as other processes.

Introduction

A significant aspect of macrophage function is their role in innate and adaptive immunity. Because of their capacity to phagocytose inert particles, bacteria or parasites, macrophages are a first line of defense against foreign invaders. Once internalized, microbes are degraded within phagolysosomes. Macrophages also send recruitment signals for and present antigens to other immune cells such as T lymphocytes. Macrophages are derived from monocytes. Monocytes arise in the bone marrow from myeloid stem cells and migrate to peripheral blood and various tissues where they differentiate into macrophages. It is estimated that a healthy adult mouse contains approximately 10....

Protocol

Ethics Statement

The protocol for animal handling was approved by our institutional Animal Ethics Committee "Conseil Scientifique du Centre de Formation et de Recherche Experimental Médico-Chirurgical" (CFREMC, Project permit 10-300122013 to Eric Ghigo) from Aix-Marseille University in accord with the rules of Décret N° 87-848 of 10/19/1987. The experiments were performed at the Faculté de Médecine de la Timone (Experimentation permit number 13.385 to Eric Ghigo).

1. Material and Culture Media Preparation

1. Sterilize two forceps, two scissors, two surgical blades, a mortar ....

Results

The aim of this method was to easily obtain large numbers of macrophages in few days. The bone marrow cell preparation is illustrated in Figure 1. Bones from the hind leg were collected and smashed in a mortar. Once the resident macrophages were removed from the bone marrow cell preparation, bone marrow cells were incubated with GM-CSF (Day 0). After 3 days, the cells, which were round and nonadherent before culture, start to differentiate into macrophages and adhere (Figure 1A). Cytomet.......

Discussion

The protocol described herein details a method to produce large numbers of BMDMs. BMDM are primary cells and have the biological function and properties of macrophages differentiated from monocytes because there are mature, in contrast to macrophage cell lines, which are immature. BMDMs may be used for genetic screening (RNAi), drug screening, functional studies, host-pathogen interaction studies and many other areas of investigation.

The procedure presented herein is very simple and does not .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM	Gibco Life Technologies	21969-035
Fetal Calf Serum	Gibco Life Technologies	10270
Penicillin/Streptomycin	Gibco Life Technologies	15070
Glutamine	Gibco Life Technologies	25030-024
PBS (10x)	Lonza	BEM515F
Red Blood Cell Lysis buffer	Sigma	R7757
Cell strainer 70 μm Nylon	BD Falcon	352350
Cell strainer 40 μm Nylon	BD Falcon	352340
50 ml tubes	any supplier	n/a
15 ml tubes	any supplier	n/a
Petri dishes (100/20 mm)	any supplier	n/a	culture treated
Petri dishes (35/10 mm)	any supplier	n/a