在聚乙烯吡咯烷酮包被的硅胶（Percoll）连续密度梯度上分离人外周血单核细胞。

Separation of human peripheral blood monocytes on continuous density gradients of polyvinylpyrrolidone-coated silica gel (Percoll).

作者信息

Brandslund I, Rasmussen J M, Fisker D, Svehag S E

出版信息

J Immunol Methods. 1982;48(2):199-211. doi: 10.1016/0022-1759(82)90194-6.

Abstract

A standardized, reproducible two-step method for separation of human peripheral blood monocytes on continuous Percoll gradients has been developed. The first step involves separation of mononuclear cell on Percoll of density 1.075 g/ml and the second step separation of monocytes from lymphocytes on a continuous Percoll gradient with a starting density of 1.075 g/ml for the formation of the gradient. The average yield during a 10 month period of daily routine use has been 74 +/- 17% (mean +/- 1 S.D.), and the average purity 63 +/- 10%. Ninety to 95% of the monocytes are viable after separation as judged from trypan blue exclusion and by ingestion of latex particles and sensitized sheep erythrocytes. The separation takes about 3 h and the total number of monocytes obtained from 40 ml of blood is in the range of 10-15 x 106. The procedure has been reliable with 3-4% separation failures, mainly due to bacterial or fungal growth in Percoll suspension or media. The contaminating cells are exclusively lymphocytes, predominantly T-lymphocytes (90-95%), when citrate is used as anticoagulant. Heparin can not be used as anticoagulant, as there appears to be a dose-dependent formation of thrombocyte aggregates which contaminate the monocytes, and result in poor separation.

摘要

已开发出一种标准化、可重复的两步法，用于在连续的 Percoll 梯度上分离人外周血单核细胞。第一步是在密度为 1.075 g/ml 的 Percoll 上分离单核细胞，第二步是在起始密度为 1.075 g/ml 的连续 Percoll 梯度上从淋巴细胞中分离单核细胞以形成梯度。在日常使用的 10 个月期间，平均产量为 74±17%（平均值±1 个标准差），平均纯度为 63±10%。根据台盼蓝排斥试验以及乳胶颗粒和致敏绵羊红细胞的摄取情况判断，90%至 95%的单核细胞在分离后仍具有活力。分离过程约需 3 小时，从 40 ml 血液中获得的单核细胞总数在 10 - 15×10⁶范围内。该方法可靠，分离失败率为 3 - 4%，主要是由于 Percoll 悬浮液或培养基中细菌或真菌生长所致。当使用柠檬酸盐作为抗凝剂时，污染细胞仅为淋巴细胞，主要是 T 淋巴细胞（90 - 95%）。肝素不能用作抗凝剂，因为似乎会形成剂量依赖性的血小板聚集体，污染单核细胞并导致分离效果不佳。