Neonatal Mouse Bone Marrow Isolation and Preparation of Bone Marrow-Derived Macrophages

Summary

This protocol describes a non-enzymatic and straightforward method for isolating 7-9-day-old neonatal mouse bone marrow cells and generating differentiated macrophages using a supernatant of L929 cells as a source of granulocyte colony-stimulating factor (M-CSF). The bone marrow-derived macrophages were further analyzed for surface antigens F4/80, CD206, CD11b, and functional competency.

Abstract

Various techniques for isolating bone marrow from adult mice have been well established. However, isolating bone marrow from neonatal mice is challenging and time-consuming, yet for some models, it is translationally relevant and necessary. This protocol describes an efficient and straightforward method for preparing bone marrow cells from 7-9-day-old pups. These cells can then be further isolated or differentiated into specific cell types of interest. Macrophages are crucial immune cells that play a major role in inflammation and infection. During development, neonatal macrophages contribute significantly to tissue remodeling. Moreover, the phenotype and functions of neonatal macrophages differ from those of their adult counterparts. This protocol also outlines the differentiation of neonatal macrophages from the isolated bone marrow cells in the presence of L929-conditioned medium. Surface markers for differentiated neonatal macrophages were assessed using flow cytometric analysis. To demonstrate functionality, the phagocytic efficiency was also tested using pH-sensitive dye-conjugated Escherichia coli.

Introduction

Bone marrow encloses both hematopoietic and mesenchymal stem cell populations that are self-renewable and can be differentiated into various cell lineages. Hematopoietic stem cells in the bone marrow give rise to myeloid and lymphoid lineages¹. Mesenchymal stem cells produce osteoblasts (bone), adipocytes (fat), or chondrocytes (cartilage)². These cells have multiple applications in the field of cell biology and tissue engineering, including gene therapy^3,4. Progenitor cells present in the bone marrow differentiate into specific cell ty....

Protocol

All procedures were approved by the West Virginia Institutional Animal Care and Use Committees and were performed following the recommendations of the Guide for the Care and Use of Laboratory Animals by the National Research Council. C57BL/6J mouse pups were used for this study. The details of all the reagents and equipment used are listed in the Table of Materials.

1. Media preparation

1. Prepare 3 mL of MEM culture media supplemented with 10% FBS, 2.......

Discussion

Research involving neonatal mouse models can present a number of challenges. Neonates have a developing immune system that is unique compared to adults⁸. As such, data generated from adult animal models should not be assumed to apply to newborns, and several published works have articulated this idea well^18,19. Therefore, neonatal-specific models and sources of cells are necessary to study the intricacies of the early-life immune response........

Materials

Name	Company	Catalog Number	Comments
40 µm strainer	Greiner	542040	Cell culture
96 well round (U) bottom plate	Thermo Scientific	12-565-65	Cell culture
Anti-mouse CD11b-BV786	BD Biosciences	740861	FACS analysis
Anti-mouse CD206-Alexa Fluor488	BD Biosciences	141709	FACS analysis
Anti-mouse F4/80-PE	BD Biosciences	565410	FACS analysis
Countess3	Thermo Scientific	TSI-C3ACC	Automated cell counter
DMEM	Hyclone	SH30022.01	Cell culture
DMSO	VWR	WN182	Cell culture
DPBS, 1x	Corning	21-031-CV	Cell culture
Escherichia coli O1:K1:H7	ATCC	11775	Infection
EVOS FL	Invitrogen	12-563-649	Cell Imaging System
FBS	Avantor	76419-584	Cell culture
FluoroBright BMDM	Thermo fisher Scientific	A1896701	Dye free culture media
Glutamine	Cytiva	SH30034.01	Cell culture
HEPES	Cytiva	SH30237.01	Cell culture
L-929	ATCC		Differentiation
LSRFortessa	Becton Dickinson		Flowcytometer
Lysotracker red DND 99	Invitrogen	L7528	Fluorescent dye
MEM	Corning	15-010-CV	Cell culture
Penicillin /streptomycin	Hyclone	SV30010	Cell culture
pHrodo green STP ester	Invitrogen	P35369	Fluorescent dye
T75 flask	Cell star	658170	Cell culture
Trypsin-EDTA	Gibco	25300120	Cell culture
Zeiss 710	Zeiss	P20GM103434	Confocal