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Journal of Immunology Research
Volume 2017, Article ID 1254792, 10 pages
https://doi.org/10.1155/2017/1254792
Research Article

The Application of Dextran Sedimentation as an Initial Step in Neutrophil Purification Promotes Their Stimulation, due to the Presence of Monocytes

1Department of Immunopathology, SA Pathology at the Women’s and Children’s Hospital, North Adelaide, SA, Australia
2School of Medicine, Robinson Research Institute and School of Biological Sciences, University of Adelaide, Adelaide, SA, Australia
3School of Pharmacy and Medical Sciences, Sansom Institute, University of South Australia, Adelaide, SA, Australia

Correspondence should be addressed to Alex Quach; ua.ude.asinu.liamym@hcauq.rednaxela

Received 18 June 2017; Revised 8 September 2017; Accepted 18 September 2017; Published 15 October 2017

Academic Editor: Nejat K. Egilmez

Copyright © 2017 Alex Quach and Antonio Ferrante. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

The purification of human neutrophils for in vitro studies is challenging as they are easily activated through ex vivo manipulations. The technique of erythrocyte sedimentation combined with density gradient centrifugation remains widely practiced and was the subject of this study. Since in the sedimentation step the leukocytes are incubated with dextran, we have raised the likelihood that cellular activation would occur with mediator release leading to neutrophil activation. By comparing the activity of neutrophils purified from whole blood by the classical 2-step method of dextran sedimentation followed by low-density Ficoll-Hypaque (1.077 g/mL) medium, and the 1-step high-density Ficoll-Hypaque (1.114 g/mL) gradient centrifugation, we found that neutrophils from the 2-step method had a significant increase in cell surface CD11b expression and CD62L shedding and a marked increase in adhesion. Decreased random migration and chemotaxis and raised baseline oxidative burst activity were also observed. The effect was not specific to dextran, as using Ficoll for erythrocyte sedimentation replicated the elevated neutrophil adherence. Through the depletion of monocytes, lymphocytes, and platelets prior to sedimentation, we deduced that monocytes were responsible for the neutrophil activation. Our findings suggest that care needs to be exercised in choosing the method of neutrophil purification for functional studies.