Preparation and surface labeling of murine eosinophils
Burgess AW., Cruise KM., Mitchell GF., Watt SM.
Eosinophilic polymorphonuclear leukocytes were isolated from the peritoneal cavity of BALB/c mice infected with the parasite Mesocestoides corti. Approximately 4x107 eosinophils (purity 50%) could be harvested from each mouse. A high yield and purity of eosinophils was obtained from the peritoneal cells of infected male BALB/c mice using density centrifugation on a gradient of slightly hypotonic colloidal silica sol (Percoll). After initial irradiation of the mice to lower the lymphocyte contamination, subsequent density gradient (and where necessary sedimentation velocity) centrifugation yielded 108 eosinophils (purity>95%) from six to eight mice. It was also possible to isolate small numbers of eosinophils (2x104 cell/minute, purity>99%) without irradiating the mice. This could be achieved by separating the density gradient purified peritoneal cells by light-scatter on a Becton-Dickinson cell sorter (FACS II). Analysis of proteins extracted from eosinophils using polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed a group of high molecular weight proteins (between 250K and 160K) which were not as distinctive in the neutrophil profile. Surface labeling was performed, before the cell separation, by using 125I and 1,3,4,6-tetrachloro-3α,6α-diphenylglycoluril. Only five 125I-labeled proteins were detected initially (all with apparent molecular weight >50,000). No 125I appeared to be associated with actin under the conditions used for surface labeling. Four of the eosinophil surface labeled proteins corresponded to surface labeled proteins on neutrophils, but the major surface component of the eosinophils (MW79,000) appeared to be smaller than the major neutrophil protein (MW 90,000).