Purification of human blood eosinophils by a combination method using anti-CD16 monoclonal antibody, immunobeads, and Nycodenz density gradient.
A simple method is described for the procurement of human blood eosinophil phenotypes by combining an anti-CD16 monoclonal antibody, immunobeads, and a non-toxic and non-ionic density gradient medium, Nycodenz. The purification depends on the removal of mononuclear cells using a 1.076/1.102 g/ml Nycodenz density gradient, partial removal of neutrophils based on different binding to plastic dishes, interaction of residual neutrophils with immunobeads via an anti-CD16 monoclonal antibody and, finally, extraction of eosinophil phenotypes by sifting the immunobeads-loaded neutrophils through an 1.080/1.102 g/ml Nycodenz density gradient. This method permits simultaneous preparation of highly purified normodense (> 1.080 g/ml) and hypodense eosinophils (< 1.080 g/ml) with reasonable chemiluminescence responses to opsonized zymosans and helminthotoxic activity to opsonized schistosomula corresponding to their own immunocytological properties.
Department of Dermatology, Kurume University School of Medicine, Japan.
M
M
Miyasato
S
S
Tsuda
N
N
Kitamura
K
K
Shirouzu
T
T
Nakama
Y
Y
Sasai
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001773:Blood Cells; D001798:Blood Proteins / Q000276:immunology; D002469:Cell Separation / Q000379:methods*; D002499:Centrifugation, Density Gradient; D003876:Dermatitis, Atopic / Q000097:blood; D047091:Eosinophil Granule Proteins; D004804:Eosinophils / Q000502:physiology; D006098:Granulocytes / Q000276:immunology; D006801:Humans; D007158:Immunologic Techniques; D008163:Luminescent Measurements; D051379:Mice; D008863:Microspheres; D017452:Receptors, IgG / Q000276:immunology; D012016:Reference Values; D012260:Ribonucleases; D012547:Schistosoma
D000911:Antibodies, Monoclonal; D001798:Blood Proteins; D047091:Eosinophil Granule Proteins; D017452:Receptors, IgG; D012260:Ribonucleases