Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2001 May:Chapter 7:7.23.1-7.23.17.
doi: 10.1002/0471142735.im0723s19.

Isolation and functional analysis of neutrophils

Robert A Clark  1 , William M Nauseef  1
Affiliations

Isolation and functional analysis of neutrophils

Robert A Clark et al. Curr Protoc Immunol. 2001 May.

Abstract

This unit describes the isolation of high-purity neutrophils (or polymorphonuclear leukocytes; PMN) and the assays that can be performed to assess their function. An assay for the in vitro study of phagocytic capacity is presented, and microbicidal assays using lysostaphin and differential centrifugation are also described. During phagocytosis or on exposure to soluble agonists, neutrophils exhibit a marked increase in oxidative metabolism which is mediated by activation of NADPH oxidase and results in the formation of superoxide anion as well as hydrogen peroxide and other reactive species. The nitroblue tetrazolium reduction slide test is presented, and is a nonquantitative screening test for the presence of reactive oxygen products. The quantitation of superoxide by the kinetic and static assays are detailed, and a "broken cell" assay is also outlined.Finally, measurement of hydrogen peroxide formation is described.

PubMed Disclaimer

References

Literature Cited
    1. Boyum, A. 1968. Separation of leukocytes from blood and bone marrow. Scand. J. Clin. Lab. Invest. Suppl. 21:77-89.
    1. Boyum, A. 1974. Separation of blood leukocytes, granulocytes and lymphocytes. Tissue Antigens 4:269-274.
    1. Borregaard, N., Heiple, J.M., Simons, E.R., and Clark, R.A. 1983. Subcellular localization of the b-cytochrome component of the human neutrophil microbicidal oxidase. Translocation during activation. J. Cell Biol. 97:52-61.
    1. Clark, R.A. and Klebanoff, S.J. 1979. Role of the myeloperoxidase-H2O2-halide system in concanavalin A-induced tumor cell killing by human neutrophils. J. Immunol. 122:2605-2610.
    1. Clark, R.A., Leidal, K.G., Pearson, D.W., and Nauseef, W.M. 1987. NADPH oxidase of human neutrophils: Subcellular localization and characterization of an arachidonate-activatable superoxide-generating system. J. Biol. Chem. 262:4065-4074.
Key References
    1. Clark et al., 1987. See above.
    1. Cohen and Chovaniec, 1978. See above.
    1. Nauseef, W.M., de Alarcon, P., Bale, J.F., and Clark, R.A. 1986. Aberrant activation and regulation of the neutrophil oxidative burst in neutrophils with Mo1 glycoprotein deficiency. J. Immunol. 137:636-642.
    1. Root, R.K., Ellman, L., and Frank, M.M. 1972. Bactericidal and opsonic properties of C4- deficient guinea pig serum. J. Immunol. 109:477-486.
    1. Root, R.K., Rosenthal, A.S., and Balestra, D.J. 1972. Abnormal bactericidal, metabolic, and lysosomal function of Chediak-Higashi syndrome leukocytes. J. Clin. Invest. 51:649-665.