Preparation of large numbers of highly purified normodense human eosinophils from leukapheresis.
Methods have been developed for the preparation of large numbers of virtually pure, normodense eosinophils from the peripheral blood of normal human volunteers by means of leukapheresis. The purification depends on the sequential removal of mononuclear cells using a one-step discontinuous density gradient, lysis of erythrocytes, enrichment of eosinophils by centrifugation through discontinuous Percoll gradients and, finally, passive selection of the eosinophils by removal of the remaining polymorphonuclear neutrophils with a monoclonal antibody to CD16. The purity of the isolated eosinophils was consistently in excess of 95%. Recovery into the normodense eosinophil fraction ranged between 10 and 87% (average 31.6 +/- 4.2) and recovery during the monoclonal antibody step averaged 80.3 +/- 8.6%. These methods have made it possible, for the first time, to isolate 2-20 x 10(7) virtually pure normodense eosinophils from the peripheral blood of a single donor for further biochemical or pharmacological experimentation.
Bach
M K
MK
Hypersensitivity Diseases Research, Upjohn Laboratories, Kalamazoo, MI 49001.
Brashler
J R
JR
Sanders
M E
ME
D000328:Adult; D000911:Antibodies, Monoclonal; D015703:Antigens, CD / Q000276:immunology; D000943:Antigens, Differentiation / Q000276:immunology; D002469:Cell Separation / Q000379:methods; D002499:Centrifugation, Density Gradient; D004804:Eosinophils / Q000166:cytology; D006801:Humans; D007937:Leukapheresis; D011205:Povidone; D011961:Receptors, Fc / Q000276:immunology; D017452:Receptors, IgG; D012822:Silicon Dioxide
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D000943:Antigens, Differentiation; D011961:Receptors, Fc; D017452:Receptors, IgG; C016039:Percoll; D012822:Silicon Dioxide; D011205:Povidone