Technical advance: autofluorescence-based sorting: rapid and nonperturbing isolation of ultrapure neutrophils to determine cytokine production.

David A Dorward,Ana L Alessandri,Adriano G Rossi,Christopher D Lucas,John A Marwick,Christopher Haslett,Fiona Rossi,Kevin Dhaliwal,Ian Dransfield

doi:10.1189/jlb.0113040

David A Dorward, Ana L Alessandri + Show 7 more

Open Access

https://doi.org/10.1189/jlb.0113040

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Abstract

The technical limitations of isolating neutrophils without contaminating leukocytes, while concurrently minimizing neutrophil activation, is a barrier to determining specific neutrophil functions. We aimed to assess the use of FACS for generating highly pure quiescent neutrophil populations in an antibody-free environment. Peripheral blood human granulocytes and murine bone marrow-derived neutrophils were isolated by discontinuous Percoll gradient and flow-sorted using FSC/SSC profiles and differences in autofluorescence. Postsort purity was assessed by morphological analysis and flow cytometry. Neutrophil activation was measured in unstimulated-unsorted and sorted cells and in response to fMLF, LTB4, and PAF by measuring shape change, CD62L, and CD11b expression; intracellular calcium flux; and chemotaxis. Cytokine production by human neutrophils was also determined. Postsort human neutrophil purity was 99.95% (sem=0.03; n=11; morphological analysis), and 99.68% were CD16(+ve) (sem=0.06; n=11), with similar results achieved for murine neutrophils. Flow sorting did not alter neutrophil activation or chemotaxis, relative to presorted cells, and no differences in response to agonists were observed. Stimulated neutrophils produced IL-1β, although to a lesser degree than CXCL8/IL-8. The exploitation of the difference in autofluorescence between neutrophils and eosinophils by FACS is a quick and effective method for generating highly purified populations for subsequent in vitro study.

Highlights

Neutrophils are key effector cells of the innate immune system that play an important role in the inflammatory cascade [1]
Whereas conventional neutrophil isolation techniques provide a convenient method of purification, several important observations with regard to neutrophil function have only been revealed through use of highly purified neutrophil populations
Cells were flow-sorted using BD FACSAria II SORP (BD Biosciences, San Jose, CA, USA) with gates set around the granulocyte population, based on characteristic FSC and SSC profiles with doublets removed on the basis of FSC-area versus FSC-height

Summary

Introduction

Neutrophils are key effector cells of the innate immune system that play an important role in the inflammatory cascade [1]. Delineating their functional, biochemical, and synthetic capabilities. Given the ease with which neutrophils can become activated, the techniques used in the isolation of neutrophils from peripheral blood can profoundly influence subsequent in vitro and in vivo function [4 –7]. Whereas use of Ficoll/Hypaque or dextran sedimentation with a subsequent discontinuous Percoll gradient is generally accepted as the isolation method least likely to activate neutrophils, purity of Ͼ95–97% is difficult to achieve [8, 9]. Subsequent purification of granulocytes by a variety of antibody cocktail/magnetic bead-based selection strategies has been described for human and murine use [10, 11]

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