Effect of isolation protocol on eosinophil function: Percoll gradients versus immunomagnetic beads.
Studies of in vitro eosinophil function are dependent on efficient and reliable methods of cell isolation. Protocols using Percoll or metrizamide density gradients have been of limited use in isolating peripheral blood eosinophils in sufficient numbers and purity from subjects with normal or only slightly elevated eosinophil counts, thereby restricting comparative studies to preparations from hypereosinophilic subjects. Recently, a method utilizing negative selection by anti-CD16 coated magnetic beads has greatly improved eosinophil isolation by dramatically increased yields and purity. However, little is known as to the differential effect of various isolation methods on the functional activity of eosinophils. In this study, eosinophils were isolated by either discontinuous multiple density Percoll gradients or anti-CD16-coated magnetic beads: several functional activities were then compared using cells obtained by the two methods of isolation. Compared with Percoll isolated eosinophils, anti-CD16 bead separated eosinophils had significantly increased baseline and stimulated LTC4 production, spontaneous O2- generation, and expression of specific cell surface markers. No significant difference was observed in the cells' in vitro survival and adhesion. Such differences may be due to the isolation of eosinophils of all densities by anti-CD16 beads, or the effect of neutrophils interacting with the beads to release eosinophil agonists or primers. Alternatively, the Percoll gradient method with the eosinophils' exposure to dextran and Ficoll-Hypaque may affect subsequent cell function. Therefore, comparison of eosinophil function between cells isolated by different protocols must be considered before concluding which is the true measure of in vivo cell function.
Department of Medicine, University of Wisconsin, Madison, USA.
J B
JB
Sedgwick
Y
Y
Shikama
M
M
Nagata
K
K
Brener
W W
WW
Busse
D000293:Adolescent; D000328:Adult; D000911:Antibodies, Monoclonal / Q000276:immunology; D001249:Asthma / Q000473:pathology; D002448:Cell Adhesion / Q000502:physiology; D002469:Cell Separation / Q000379:methods*; D002499:Centrifugation, Density Gradient; D003102:Colloids; D004730:Endothelium, Vascular / Q000166:cytology; D004804:Eosinophils / Q000276:immunology* / Q000502:physiology; D005434:Flow Cytometry; D006801:Humans; D018189:Immunomagnetic Separation; D017997:Leukotriene C4 / Q000096:biosynthesis; D008875:Middle Aged; D011205:Povidone; D017452:Receptors, IgG / Q000276:immunology; D006255:Rhinitis, Allergic, Seasonal / Q000473:pathology; D012822:Silicon Dioxide; D014471:Umbilical Veins / Q000166:cytology
D000911:Antibodies, Monoclonal; D003102:Colloids; D017452:Receptors, IgG; D017997:Leukotriene C4; C016039:Percoll; D012822:Silicon Dioxide; D011205:Povidone