Rapid Immunomagnetic Negative Enrichment of Neutrophil Granulocytes from Murine Bone Marrow for Functional Studies In Vitro and In Vivo

Mike Hasenberg,Julia Achilles,Katrin Borucki,Linda Männ,Monika Riek-Burchardt,Anja Köhler,Susanne Bonifatius,Kathleen Baumgart,Burkhart Schraven,Matthias Gunzer,Dominik Hartl

doi:10.1371/journal.pone.0017314

Abstract

Polymorphonuclear neutrophils (PMN) mediate early immunity to infection but can also cause host damage if their effector functions are not controlled. Their lack or dysfunction is associated with severe health problems and thus the analysis of PMN physiology is a central issue. One prerequisite for PMN analysis is the availability of purified cells from primary organs. While human PMN are easily isolated from peripheral blood, this approach is less suitable for mice due to limited availability of blood. Instead, bone marrow (BM) is an easily available reservoir of murine PMN, but methods to obtain pure cells from BM are limited. We have developed a novel protocol allowing the isolation of highly pure untouched PMN from murine BM by negative immunomagnetic isolation using a complex antibody cocktail. The protocol is simple and fast (∼1 h), has a high yield (5–10*106 PMN per animal) and provides a purity of cells equivalent to positive selection (>80%). Most importantly, cells obtained by this method are non-activated and remain fully functional in vitro or after adoptive transfer into recipient animals. This method should thus greatly facilitate the study of primary murine PMN in vitro and in vivo.

Highlights

Polymorphonuclear neutrophils (PMN) are the most abundant and, arguably, most important cell type of the innate immune system
To establish a protocol that would allow the negative isolation of murine neutrophils from bone marrow we first had to identify good candidate surface markers which are present on marrow resident cells but absent from neutrophils
The majority of neutrophils in this tissue is of the Gr-1high phenotype and represents mature neutrophils displaying the same expression profile of classical activation markers upon stimulation as cells isolated by positive selection

Summary

Introduction

Polymorphonuclear neutrophils (PMN) are the most abundant and, arguably, most important cell type of the innate immune system. By non specific mechanisms such as phagocytosis and extracellular killing via toxic agents they mediate the first line of defense against infection. This early pathogen control provides the relatively slow adaptive immune system with a time window allowing to develop tailored responses that can sterilize the body from the infection and provide longterm protection [1]. Despite many decades of research still new principal defense mechanisms of PMN are being uncovered such as the formation of extracellular DNA-nets [2,3]. PMN physiology remains a central and actively investigated topic of immunological and hematological research

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Conclusion