Flow Cytometry Analysis of Murine Bone Marrow Hematopoietic Stem and Progenitor Cells and Stromal Niche Cells

Summary

Here, we describe a simple protocol for the isolation and staining of murine bone marrow cells to phenotype hemopoietic stem and progenitor cells along with the supporting niche endothelial and mesenchymal stem cells. A method to enrich cells located in endosteal and central bone marrow areas is also included.

Abstract

The bone marrow (BM) is the soft tissue found within bones where hematopoiesis, the process by which new blood cells are generated, primarily occurs. As such, it contains hematopoietic stem and progenitor cells (HSPCs), as well as supporting stromal cells that contribute to the maintenance and regulation of HSPCs. Hematological and other BM disorders disrupt hematopoiesis by affecting hematopoietic cells directly and/or through the alteration of the BM niche. Here, we describe a method to study hematopoiesis in health and malignancy through the phenotypic analysis of murine BM HSPCs and stromal niche populations by flow cytometry. Our method details the required steps to enrich BM cells in endosteal and central BM fractions, as well as the appropriate gating strategies to identify the two key niche cell types involved in HSPC regulation, endothelial cells and mesenchymal stem cells. The phenotypic analysis proposed here may be combined with mouse mutants, disease models, and functional assays to characterize the HSPC compartment and its niche.

Introduction

Flow cytometry is an invaluable method to characterize and prospectively isolate immune and hematopoietic cells. It is also increasingly being used to analyze stromal and epithelial populations of different tissues. The hematopoietic stem cell (HSC) has unique properties of self-renewal and multipotency. In adult mammals, HSCs primarily reside in the bone marrow (BM), where they receive quiescence and survival signals from the surrounding microenvironment or niche¹. HSCs are formally defined according to functional assays². Nevertheless, several landmark papers have shown the usefulness of flow cytometry to identify HSCs....

Protocol

The animals used in this protocol were housed at the i3S animal facility under specific pathogen-free conditions in a 12 h light-dark cycle and temperature-controlled environment. Free access to standard rodent chow and water was provided. All the animals received humane care according to the criteria outlined by the Federation of European Laboratory Animal Science Associations for the care and handling of laboratory animals (EU Directive 2010/63/EU). The experimental procedure performed on the animals (euthanasia) was a.......

Discussion

While the protocol described is simple and easy to perform, special attention should be brought to specific steps. For example, when obtaining flushed BM (step 5.2), the volume or number of times indicated to pass PBS 2% FBS through the inside of the central part of the bone should not be exceeded, as this might result in significant contamination of the flushed sample by endosteal cell populations.

Alterations to the protocol can be made to facilitate its execution by the investigator. In sam.......

Materials

Name	Company	Catalog Number	Comments
Alexa Fluor 647 anti-mouse CD54/ICAM-1 antibody	BioLegend	116114
APC Streptavidin	BioLegend	405207
APC/Cyanine7 anti-mouse CD117 (c-kit) antibody	BioLegend	105826
APC/Cyanine7 anti-mouse CD45 antibody	BioLegend	103116
APC/Cyanine7 anti-mouse TER-119/erythroid cells antibody	BioLegend	116223
Biotin anti-mouse CD3ε antibody	BioLegend	100304
Biotin anti-mouse CD4 antibody	BioLegend	100404
Biotin anti-mouse CD8a antibody	BioLegend	100704
Biotin anti-mouse Ly-6G/Ly-6C (Gr-1) antibody	BioLegend	108404
Biotin anti-mouse TER-119/erythroid cells antibody	BioLegend	116204
Biotin anti-mouse/human CD11b antibody	BioLegend	101204
Biotin anti-mouse/human CD45R/B220 antibody	BioLegend	103204
Brilliant Violet 510 anti-mouse CD150 (SLAM) antibody	BioLegend	115929
Calibrite 2 Color Beads	BD Biosciences	349502
Collagenase IV	Merck Life Science	C1889
Dispase II	Merck Life Science	D4693
Fetal Bovine Serum, qualified, heat inactivated, E.U.-approved, South America Origin	ThermoFisher Scientific	10500064
Hanks' Balanced Salt Solution (HBSS)	ThermoFisher Scientific	14175095
Mouse Leptin R Biotinylated Antibody	R&D systems	BAF497
NucBlue Fixed Cell Reagent (DAPI)	ThermoFisher Scientific	R37606	DAPI reagent
PE anti-mouse endomucin antibody	ThermoFisher Scientific	12-5851-82
PE anti-mouse Flk2 (CD135)	ThermoFisher Scientific	12-1351-82
PE/Cyanine7 anti-mouse CD31 antibody	BioLegend	102524
PE/Cyanine7 anti-mouse CD48 antibody	BioLegend	103424
PerCP anti-mouse Ly-6A/E (Sca-1) antibody	BioLegend	108122
Phosphate-buffered saline (PBS) tablets	Merck Life Science	P4417
Purified anti-mouse CD16/32 antibody	BioLegend	101302
RBC lysis buffer 10x	BioLegend	420302
Zombie Violet Fixable Viability Dye	BioLegend	423114	fluorescent dye