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Neutrophil Isolation from Nonhuman Species

  • Daniel W. Siemsen
  • Natalia Malachowa
  • Igor A. Schepetkin
  • Adeline R. Whitney
  • Liliya N. Kirpotina
  • Benfang Lei
  • Frank R. DeLeo
  • Mark T. Quinn
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1124)

Abstract

The development of new advances in the understanding of neutrophil biochemistry requires effective procedures for isolating purified neutrophil populations. Although methods for human neutrophil isolation are now standard, similar procedures for isolating neutrophils from many of the nonhuman species used to model human diseases are not as well developed. Since neutrophils are reactive cells, the method of isolation is extremely important to avoid isolation technique-induced alterations in cell function. We present methods here for reproducibly isolating highly purified neutrophils from large animals (bovine, equine, ovine), small animals (murine and rabbit), and nonhuman primates (cynomolgus macaques), and describe optimized details for obtaining the highest cell purity, yield, and viability. We also describe methods to verify phagocytic capacity in the purified cell populations using a flow cytometry-based phagocytosis assay.

Keywords

Inflammation Phagocytosis Large animal model Granulocyte Polymorphonuclear leukocyte Cell isolation Flow cytometry Blood Bone marrow 

Notes

Acknowledgements

This work was supported in part by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences, National Institutes of Health under grant number GM103500 (D.S., I.A.S., L.K.N., B.L., M.Q.), the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health (N.M., A.R.W., F.R.D.), and the Montana State University Agricultural Experimental Station.

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Copyright information

© Springer Science+Business Media, LLC 2014

Authors and Affiliations

  • Daniel W. Siemsen
    • 1
  • Natalia Malachowa
    • 2
  • Igor A. Schepetkin
    • 1
  • Adeline R. Whitney
    • 2
  • Liliya N. Kirpotina
    • 1
  • Benfang Lei
    • 1
  • Frank R. DeLeo
    • 2
  • Mark T. Quinn
    • 1
  1. 1.Department of Immunology and Infectious DiseasesMontana State UniversityBozemanUSA
  2. 2.Laboratory of Human Bacterial Pathogenesis, Rocky Mountain LaboratoriesNational Institute of Allergy and Infectious Diseases, National Institutes of HealthHamiltonUSA

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