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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Wiles S, Hanage WP, Frankel G et al (2006) Modelling infectious disease – time to think outside the box? Nat Rev Microbiol 4: 307–312
Casal M, Haskins M (2006) Large animal models and gene therapy. Eur J Hum Genet 14:266–272
Styrt B (1989) Species variation in neutrophil biochemistry and function. J Leukoc Biol 46: 63–74
Glasser L, Fiederlein RL (1990) The effect of various cell separation procedures on assays of neutrophil function. A critical appraisal. Am J Clin Pathol 93:662–669
Watson F, Robinson JJ, Edwards SW (1992) Neutrophil function in whole blood and after purification – changes in receptor expression, oxidase activity and responsiveness to cytokines. Biosci Rep 12:123–133
Forsyth KD, Levinsky RJ (1990) Preparative procedures of cooling and re-warming increase leukocyte integrin expression and function on neutrophils. J Immunol Methods 128: 159–163
Macey MG, Jiang XP, Veys P et al (1992) Expression of functional antigens on neutrophils. Effects of preparation. J Immunol Methods 149:37–42
Alvarez-Larrán A, Toll T, Rives S et al (2005) Assessment of neutrophil activation in whole blood by flow cytometry. Clin Lab Haematol 27:41–46
Pycock JF, Allen WE, Morris TH (1987) Rapid, single-step isolation of equine neutrophils on a discontinuous Percoll density gradient. Res Vet Sci 42:411–412
Lowell CA, Fumagalli L, Berton G (1996) Deficiency of Src family kinases p59/61hck and p58c-fgr results in defective adhesion-dependent neutrophil functions. J Cell Biol 133:895–910
Woldehiwet Z, Scaife H, Hart CA et al (2003) Purification of ovine neutrophils and eosinophils: anaplasma phagocytophilum affects neutrophil density. J Comp Pathol 128:277–282
White-Owen C, Alexander JW, Sramkoski RM et al (1992) Rapid whole-blood microassay using flow cytometry for measuring neutrophil phagocytosis. J Clin Microbiol 30: 2071–2076
Doerschuk CM, Allard MF, Martin BA et al (1987) Marginated pool of neutrophils in rabbit lungs. J Appl Physiol 63:1806–1815
DeLeo FR, Renee J, Mccormick S et al (1998) Neutrophils exposed to bacterial lipopolysaccharide upregulate NADPH oxidase assembly. J Clin Invest 101:455–463
Siemsen DW, Schepetkin IA, Kirpotina LN, Lei B, Quinn MT (2007) Neutrophil isolation from non-human species. Methods Mol Biol 412:21–34
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.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Siemsen, D.W. et al. (2014). Neutrophil Isolation from Nonhuman Species. In: Quinn, M., DeLeo, F. (eds) Neutrophil Methods and Protocols. Methods in Molecular Biology, vol 1124. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-845-4_3
Download citation
DOI: https://doi.org/10.1007/978-1-62703-845-4_3
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-844-7
Online ISBN: 978-1-62703-845-4
eBook Packages: Springer Protocols