Ammonium chloride exposure inhibits cytokine-mediated eosinophil survival.
To study human eosinophils, their efficient purification from peripheral blood is crucial. Although a number of purification procedures, including discontinuous Percoll and metrizamide density gradient centrifugation, have been used, it has been difficult to isolate eosinophils from normal donors with consistently high yields and purities. Recently, a new isolation technique called magnetic cell separation system (MACS) was reported. To evaluate this procedure, we isolated eosinophils from human peripheral blood using either MACS or the standard discontinuous Percoll density methods, and compared cellular viability, morphology, and response to degranulation stimuli. MACS gave a higher yield of eosinophils than Percoll density centrifugation; for example, 6.6 +/- 1.1 x 10(6) eosinophils were isolated from 20 ml of blood by MACS compared to 6.4 +/- 2.4 x 10(6) from 120 ml by Percoll density gradient. Further, the purity of eosinophils isolated by MACS was 97.1 +/- 0.5% (X +/- SEM) compared to 77.8 +/- 2.9% with Percoll. As part of the MACS protocol, erythrocytes are lysed with either 155 mM ammonium chloride or hypotonic lysis. With 155 mM ammonium chloride treatment, the eosinophils showed a striking reduction in cytokine mediated survival due to interleukin (IL)-3, IL-5 and granulocyte-macrophage colony-stimulating factor (GM-CSF), marked morphologic abnormalities and a reduced degranulation response. With hypotonic lysis, no differences were observed in survival and morphology between eosinophils purified by MACS and Percoll methods; the degranulation responses to stimuli were essentially the same between the two methods. Taken together, these observations suggest that the exposure of eosinophils to 155 mM ammonium chloride results in cellular damage. Therefore, MACS with hypotonic lysis is a useful technique to isolate eosinophils for biological study.
Ide
M
M
Department of Immunology, Mayo Clinic, Rochester, MN 55905.
Weiler
D
D
Kita
H
H
Gleich
G J
GJ
D000643:Ammonium Chloride / Q000494:pharmacology*; D015550:Cell Degranulation / Q000502:physiology*; D002469:Cell Separation / Q000379:methods; D002470:Cell Survival / Q000187:drug effects; D002499:Centrifugation, Density Gradient; D016207:Cytokines / Q000037:antagonists & inhibitors*; D004804:Eosinophils / Q000187:drug effects* / Q000502:physiology / Q000648:ultrastructure; D006801:Humans; D018189:Immunomagnetic Separation; D007958:Leukocyte Count; D011205:Povidone; D012822:Silicon Dioxide
D016207:Cytokines; D000643:Ammonium Chloride; C016039:Percoll; D012822:Silicon Dioxide; D011205:Povidone