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Isolation of Mouse Neutrophils

Yi Luo¹, Martin E. Dorf¹

¹Harvard Medical School, Boston, Massachusetts

Publication Name:

Current Protocols in Immunology

Unit Number:

Unit 3.20

DOI:

10.1002/0471142735.im0320s22

Online Posting Date:

May, 2001

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Abstract

Neutrophils are widely used in research to elucidate the mechanisms of inflammation, including such processes as cell adhesion, chemotaxis, superoxide release, production of reactive nitrogen intermediates, and granule exocytosis. This unit presents a protocol for the induction of an enriched exudate of polymorphonuclear leukocytes (PMN) in the peritoneal cavity of mice. Neutrophils are then isolated using continuous gradient centrifugation. This is followed by a protocol for isolating neutrophils from peripheral blood. An alternate method for the purification of PMN from peritoneal exudate cells or peripheral blood by Histopaque density gradient centrifugation is also provided. The cell purification protocol can be modified to obtain nonelicited neutrophils from peritoneal fluid.

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Unit Introduction
Basic Protocol 1: Isolation of Neutrophils from Peritoneal Fluid
Basic Protocol 2: Isolation of Neutrophils from Peripheral Blood
Alternate Protocol: Isolation of Neutrophils from Peritoneal Fluid or Peripheral Blood
Reagents and Solutions
Commentary
Bibliography
Figures

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Materials

Basic Protocol 1: Isolation of Neutrophils from Peritoneal Fluid

Materials

Casein solution (see recipe)
Donor mice (2- to 4-month-old male mice of any conventional strain)
Harvest solution: 0.02% EDTA in 1× PBS (see recipe for 10×), filter-sterilized through 0.2-µm filter (room temperature)
70% ethanol
1× PBS (see recipe for 10×)
Percoll gradient solution (see recipe), room temperature
Medium to be used with neutrophils in subsequent experimental procedures, room temperature
Medium to be used with neutrophils in subsequent experimental procedures, containing 50% (v/v) FBS, room temperature
Diff-Quik staining solutions (Baxter)
10-ml syringes and 21-G needles
Forceps
Small, straight surgical scissors
15- or 50-ml conical polypropylene centrifuge tubes
Tabletop centrifuge
10-ml ultracentrifuge tubes (Beckman or equivalent)
Sorvall RC70 ultracentrifuge with T-1270 fixed-angle rotor (or equivalent)
Cytospin cytocentrifuge including chambers and filter cards (Shandon/Lipshaw)
Microscope slides appropriate for use with Cytospin centrifuge
Additional reagents and equipment for intraperitoneal injection (unit 1.6 ), euthanasia of mice (unit 1.8), harvesting peritoneal exudate cells (unit 14.1), and counting viable cells by trypan blue exclusion (appendix 3B)

Basic Protocol 2: Isolation of Neutrophils from Peripheral Blood

Materials

Naive mice
Methoxyflurane (Metofane)
Heparin
1× PBS (see recipe for 10×)
1-ml syringe with 20- to 22-G needle
12 × 75–mm glass tube
Tabletop centrifuge
Additional reagents and equipment for methoxyflurane anesthesia of mice (unit 1.4), cardiac puncture of mice (unit 1.7), euthanasia of mice (unit 1.8), counting cells using a hemacytometer (appendix 3A), and isolation of neutrophils by density gradient centrifugation (as for peritoneal fluid; see Basic Protocol 1, steps to )

Alternate Protocol: Isolation of Neutrophils from Peritoneal Fluid or Peripheral Blood

Additional Materials (also see Basic Protocols 1 and 2)

Histopaque-1119 and -1077 (polysucrose/sodium diatrizoate, densities 1.1119 and 1.077, respectively; store at 4°C; Sigma)
15-ml polypropylene centrifuge tubes

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Figures

Figure 3.20.1

Separation of casein-induced peritoneal neutrophils following purification by Percoll density gradient centrifugation. The faint upper band contains primarily lymphocytes and macrophages (L,M), the broad middle band includes neutrophils and other granulocytes (polymorphonuclear leukocytes; PMN), and the bottom band includes erythrocytes (E).

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Literature Cited

Literature Cited
	Devi, S., Laning, J., Luo, Y., and Dorf, M.E. 1995. Biologic activities of the -chemokine TCA3 on neutrophils and macrophages. J. Immunol. 154:5376-5383.
	Tansho, S., Abe, S., and Yamaguchi, H. 1994. Inhibition of Candida albicans growth by murine peritoneal neutrophils and augmentation of the inhibitory activity by bacterial lipopolysaccharide and cytokines. Microbiol. Immunol. 38:379-383.
	Vassalli, J-D., Granelli-Piperno, A., Griscelli, C., and Reich, E. 1978. Specific protease deficiency in polymorphonuclear leukocytes of Chediak-Higashi syndrome and beige mice. J. Exp. Med. 147:1285-1290.
	Voncken, J.W., van Schaick, H., Kaartinen, V., Deemer, K., Coates, T., Landing, B., Pattengale, P., Dorseuil, O., Bokoch, G.M., Groffen, J., and Heisterkamp, N. 1995. Increased neutrophil respiratory burst in bcr-null mutants. Cell. 80:719-728.
	Watt, Burgess, A.W., and Metcalf, D. 1979. Isolation and surface labeling of murine polymorphonuclear neutrophils. J. Cell Physiol. 100:1-22.
	Yoshinaga, M., Nakamura, S., and Hayashi, H. 1975. Interaction between lymphocytes and inflammatory exudate cells. I. Enhancement of thymocyte response to PHA by product(s) of polymorphonuclear leukocytes and macrophages. J. Immunol. 115:533-538.
Key Reference
	Watt et al., S.M. 1979. See above.
Original description of this procedure.