Ide M, Weiler D, Kita H, Gleich G J
Department of Immunology, Mayo Clinic, Rochester, MN 55905.
J Immunol Methods. 1994 Feb 10;168(2):187-96. doi: 10.1016/0022-1759(94)90054-x.
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.
为了研究人类嗜酸性粒细胞，从外周血中高效纯化它们至关重要。尽管已经使用了多种纯化程序，包括不连续的Percoll和甲泛葡胺密度梯度离心法，但一直难以从正常供体中分离出产量和纯度始终很高的嗜酸性粒细胞。最近，报道了一种称为磁性细胞分离系统（MACS）的新分离技术。为了评估该程序，我们使用MACS或标准的不连续Percoll密度方法从人外周血中分离嗜酸性粒细胞，并比较细胞活力、形态以及对脱颗粒刺激的反应。MACS分离出的嗜酸性粒细胞产量高于Percoll密度离心法；例如，通过MACS从20毫升血液中分离出6.6±1.1×10⁶个嗜酸性粒细胞，而通过Percoll密度梯度从120毫升血液中分离出6.4±2.4×10⁶个。此外，通过MACS分离的嗜酸性粒细胞纯度为97.1±0.5%（X±SEM），而Percoll法为77.8±2.9%。作为MACS方案的一部分，红细胞用155 mM氯化铵或低渗裂解液裂解。用155 mM氯化铵处理后，嗜酸性粒细胞因白细胞介素（IL）-3、IL-5和粒细胞-巨噬细胞集落刺激因子（GM-CSF）介导的存活显著降低，形态出现明显异常，脱颗粒反应减弱。用低渗裂解时，通过MACS和Percoll方法纯化的嗜酸性粒细胞在存活和形态上没有差异；两种方法对刺激的脱颗粒反应基本相同。综上所述，这些观察结果表明嗜酸性粒细胞暴露于155 mM氯化铵会导致细胞损伤。因此，采用低渗裂解的MACS是一种用于生物学研究分离嗜酸性粒细胞的有用技术。
