Bøyum A, Løvhaug D, Tresland L, Nordlie E M
Norwegian Defence Research Establishment, Division for Environmental Toxicology, Kjeller.
Scand J Immunol. 1991 Dec;34(6):697-712. doi: 10.1111/j.1365-3083.1991.tb01594.x.
This paper briefly reviews commonly used procedures for separation of mononuclear cells (MNC) and granulocytes from human blood with X-ray contrast media as gradient material, and also presents new and modified procedures for leucocyte preparation. Standard techniques for human blood do not always yield satisfactory results with blood from other species. In general pure MNC are easily obtained (top fraction), but often the granulocyte fraction has a low purity, due to contamination with MNC that move to the bottom during centrifugation and contaminate the granulocyte suspension. Obviously the density distribution of MNC differs between species. However, the separation can be improved by fine adjustment of gradient medium osmolality. For this purpose we have used Nycodenz, a non-ionic X-ray contrast medium. A favourable property of Nycodenz solutions is that the osmolality and density can easily be varied over a broad range. The cells react promptly to a change of medium osmolality. In hypertonic medium the cells expel water, shrink, their density increases and they sediment faster, in spite of a smaller radius. Further, the cells may pass what was initially a density barrier. A hypotonic environment has the opposite effect. In the present work we were able to show that a slight change of medium osmolality clearly improved different techniques for separation of leucocyte subgroups. For instance, the Isopaque-Ficoll (IF) technique consistently yielded MNC and granulocytes of high purity with human blood. However, with blood from rabbits, rats and mice the granulocyte suspensions were contaminated by 40-60% MNC. By utilizing Nycodenz, and lowering the osmolality by 10-12 per cent (at constant density--1.077 g/ml) we obtained satisfactory separation of MNC as well as granulocytes with blood from these species. A problem in the routine separation of granulocytes (IF) is a high contamination of erythrocytes (2-5 per cell) in the granulocyte suspension. With a two-layer technique with Nycodenz solutions of different densities it was possible to separate granulocytes almost devoid of erythrocytes, after proper adjustment of osmolality. By appropriate combination of density and osmolality, Nycodenz was a suitable gradient material in other separation procedures as well, e.g. the separation of monocytes and mast cells. To facilitate the use of Nycodenz as a versatile gradient material, a computer program providing recipes for various Nycodenz solutions is included as an appendix.
本文简要回顾了以X射线造影剂为梯度材料从人血中分离单核细胞（MNC）和粒细胞的常用方法，并介绍了白细胞制备的新方法和改进方法。人类血液的标准技术对其他物种的血液并不总是能产生令人满意的结果。一般来说，纯MNC很容易获得（上层部分），但粒细胞部分的纯度往往较低，这是因为在离心过程中移至底部的MNC会污染粒细胞悬浮液。显然，不同物种之间MNC的密度分布有所不同。然而，可以通过精细调整梯度介质的渗透压来改善分离效果。为此，我们使用了非离子型X射线造影剂Nycodenz。Nycodenz溶液的一个有利特性是其渗透压和密度可以在很宽的范围内轻松变化。细胞对介质渗透压的变化反应迅速。在高渗介质中，细胞排出水分，收缩，其密度增加，尽管半径变小，但沉降速度更快。此外，细胞可能会越过最初的密度屏障。低渗环境则有相反的效果。在本研究中，我们能够证明介质渗透压的轻微变化明显改善了白细胞亚群分离的不同技术。例如，异泛影葡胺-聚蔗糖（IF）技术对人血始终能产生高纯度的MNC和粒细胞。然而，对于兔、大鼠和小鼠的血液，粒细胞悬浮液被40%-60%的MNC污染。通过使用Nycodenz，并将渗透压降低10%-12%（在恒定密度——1.077 g/ml），我们用这些物种的血液获得了MNC和粒细胞的满意分离效果。粒细胞常规分离（IF）中的一个问题是粒细胞悬浮液中红细胞的高污染率（每细胞2%-5%）。采用两层技术，使用不同密度的Nycodenz溶液，在适当调整渗透压后，可以分离出几乎不含红细胞的粒细胞。通过适当组合密度和渗透压，Nycodenz在其他分离程序中也是一种合适的梯度材料，例如单核细胞和肥大细胞的分离。为便于将Nycodenz用作通用梯度材料，附录中包含一个计算机程序，提供各种Nycodenz溶液的配方。
