Davies D E, Lloyd J B
Department of Biological Sciences, University of Keele, Staffordshire, U.K.
J Immunol Methods. 1989 Mar 10;118(1):9-16. doi: 10.1016/0022-1759(89)90046-x.
Improved density-gradient methods, using Percoll or Nycodenz, have recently been introduced for the isolation of human monocytes, but the capacity of cells thus isolated to differentiate into macrophages has not been systematically studied. We have compared Percoll and Nycodenz methods for the isolation of monocytes from human blood. The Nycodenz method yielded a monocyte population of high purity, but the yield was low. The Percoll method gave almost quantitative yield of monocytes, and the contaminating cells, mostly lymphocytes, were readily washed away after allowing the monocytes to adhere to a plastic surface. The Percoll method was then successfully scaled up, providing a simple method to obtain the monocytes from 180 ml blood. These monocytes were maintained in culture and their capacity to mature into macrophages was studied, using the following criteria: increase in cell size and protein content, increase in specific activity of hexosaminidase, differential hexosaminidase release on exposure to opsonized zymosan and unopsonized polystyrene beads, loss of peroxidase activity, and development of fluoride-insensitivity by the cells' cytochemically demonstrable esterase. The cells also displayed morphological changes typical of the monocyte-to-macrophage transition. The procedures reported constitute a simple and reliable method for the production of human macrophages in increased yield.
最近引入了使用Percoll或Nycodenz的改进密度梯度方法来分离人单核细胞，但尚未系统研究由此分离的细胞分化为巨噬细胞的能力。我们比较了Percoll和Nycodenz方法从人血中分离单核细胞的效果。Nycodenz方法产生的单核细胞群体纯度高，但产量低。Percoll方法能获得几乎定量的单核细胞产量，并且在使单核细胞粘附于塑料表面后，污染细胞（主要是淋巴细胞）很容易被洗去。然后成功扩大了Percoll方法的规模，提供了一种从180毫升血液中获取单核细胞的简单方法。将这些单核细胞进行培养，并使用以下标准研究它们成熟为巨噬细胞的能力：细胞大小和蛋白质含量增加、己糖胺酶比活性增加、暴露于调理酵母聚糖和未调理聚苯乙烯珠时己糖胺酶差异释放、过氧化物酶活性丧失以及细胞化学可显示的酯酶对氟化物不敏感的发展。细胞还表现出单核细胞向巨噬细胞转变的典型形态变化。所报道的程序构成了一种简单可靠的方法，可提高人巨噬细胞的产量。
