A novel method for isolation of neutrophils from murine blood using negative immunomagnetic separation

Am J Pathol. 2001 Aug;159(2):473-81. doi: 10.1016/S0002-9440(10)61719-1.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CD18 Antigens / blood
  • Cell Survival
  • Chemotaxis, Leukocyte
  • Flow Cytometry
  • Immunomagnetic Separation / methods*
  • In Vitro Techniques
  • L-Selectin / blood
  • Leukocyte Transfusion
  • Mice
  • Mice, Inbred BALB C
  • N-Formylmethionine Leucyl-Phenylalanine / pharmacology
  • Neutrophils / cytology*
  • Neutrophils / drug effects
  • Neutrophils / physiology*
  • Reproducibility of Results
  • Tetradecanoylphorbol Acetate / pharmacology
  • Venules / physiology

Substances

  • CD18 Antigens
  • L-Selectin
  • N-Formylmethionine Leucyl-Phenylalanine
  • Tetradecanoylphorbol Acetate