Abstract
Advances in the understanding of neutrophil biochemistry require the development of 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. Because 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 (cow, horse, sheep) and small (mouse, rabbit) animal models 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.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Wiles, S., Hanage, W. P., Frankel, G., and Robertson, B. (2006) Modelling infectious disease—time to think outside the box? Nat. Rev. Microbiol. 4, 307–312.
Casal, M. and 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. and Fiederlein, R. L. (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, J. J., and Edwards, S. W. (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, K. D. and Levinsky, R. J. (1990) Preparative procedures of cooling and re-warming increase leukocyte integrin expression and function on neutrophils. J. Immunol. Methods 128, 159–163.
Macey, M. G., Jiang, X. P., Veys, P., McCarthy, D., and Newland, A. C. (1992) Expression of functional antigens on neutrophils. Effects of preparation. J. Immunol. Methods 149, 37–42.
Alvarez-Larrán, A., Toll, T., Rives, S., and Estella, J. (2005) Assessment of neutrophil activation in whole blood by flow cytometry. Clin. Lab. Haematol. 27, 41–46.
Pycock, J. F., Allen, W. E., and Morris, T. H. (1987) Rapid, single-step isolation of equine neutrophils on a discontinuous Percoll density gradient. Res. Vet. Sci. 42, 411–412.
Lowell, C. A., Fumagalli, L., and 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, C. A., and Edwards, S. W. (2003) Purification of ovine neutrophils and eosinophils: Anaplasma phagocytophilum affects neutrophil density. J. Comp. Pathol. 128, 277–282.
White-Owen, C., Alexander, J. W., Sramkoski, R. M., and Babcock, G. F. (1992) Rapid whole-blood microassay using flow cytometry for measuring neutrophil phagocytosis. J. Clin. Microbiol. 30, 2071–2076.
Doerschuk, C. M., Allard, M. F., Martin, B. A., MacKenzie, A., Autor, A. P., and Hogg, J. C. (1987) Marginated pool of neutrophils in rabbit lungs. J. Appl. Physiol. 63, 1806–1815.
DeLeo, F. R., Renee, J., Mccormick, S., et al. (1998) Neutrophils exposed to bacterial lipopolysaccharide upregulate NADPH oxidase assembly. J. Clin. Invest. 101, 455–463.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2007 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Siemsen, D.W., Schepetkin, I.A., Kirpotina, L.N., Lei, B., Quinn, M.T. (2007). Neutrophil Isolation From Nonhuman Species. In: Quinn, M.T., DeLeo, F.R., Bokoch, G.M. (eds) Neutrophil Methods and Protocols. Methods in Molecular Biology™, vol 412. Humana Press. https://doi.org/10.1007/978-1-59745-467-4_3
Download citation
DOI: https://doi.org/10.1007/978-1-59745-467-4_3
Publisher Name: Humana Press
Print ISBN: 978-1-58829-788-4
Online ISBN: 978-1-59745-467-4
eBook Packages: Springer Protocols