title: A novel method for isolation of neutrophils from murine blood using negative immunomagnetic separation.
abstract: Inappropriate neutrophil activation has been implicated in the pathology of several clinically important inflammatory conditions. Although murine models are extensively used in the investigation of such pathological processes, a reliable method by which viable, quiescent neutrophils can be isolated from murine blood has not been developed. Here we describe a novel method based on negative immunomagnetic separation, which yields highly pure populations of murine neutrophils. Blood is incubated with a cocktail of antibodies against specific cell markers on unwanted cells, and then with secondary antibody-coated magnetic beads. After running the preparation through a column within a magnetic field, labeled cells are retained, and a neutrophil-rich effluent is collected. This method yields a >95% pure suspension of >97% viable neutrophils, recovering approximately 70% of neutrophils from whole blood. Flow cytometric analysis shows little difference in surface L-selectin and CD18 expression on isolated neutrophils compared with neutrophils in whole blood, indicating that neutrophils are minimally activated bythe isolation process. Stimulation with phorbol 12-myristate 13-acetate (PMA) reduced L-selectin andincreased CD18 expression. Isolated neutrophilsmigrate under agarose in response to fMLP, and fluorescently labeled neutrophils transfused into recipient mice interact with postcapillary venules in a manner comparable to endogenous leukocytes. These findings show that neutrophils isolated using this method can be used for inflammatory studies in vitro and in vivo.
authors.affiliation: Cardiovascular Research Group, Division of Clinical Sciences North, University of Sheffield, Sheffield S5 7AU, United Kingdom.
authors.forename: M J
authors.initials: MJ
authors.lastname: Cotter
authors.forename: K E
authors.initials: KE
authors.lastname: Norman
authors.forename: P G
authors.initials: PG
authors.lastname: Hellewell
authors.forename: V C
authors.initials: VC
authors.lastname: Ridger
mesh_terms: D000818:Animals; D018821:CD18 Antigens / Q000097:blood; D002470:Cell Survival; D002634:Chemotaxis, Leukocyte; D005434:Flow Cytometry; D018189:Immunomagnetic Separation / Q000379:methods*; D066298:In Vitro Techniques; D019041:L-Selectin / Q000097:blood; D017708:Leukocyte Transfusion; D051379:Mice; D008807:Mice, Inbred BALB C; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000166:cytology* / Q000187:drug effects / Q000502:physiology*; D015203:Reproducibility of Results; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014699:Venules / Q000502:physiology
chemical_list: D018821:CD18 Antigens; D019041:L-Selectin; D009240:N-Formylmethionine Leucyl-Phenylalanine; D013755:Tetradecanoylphorbol Acetate