Pertoft H
Department of Medical Biochemistry and Microbiology, Unit of Biochemistry, University of Uppsala BMC, Box 575, SE-75123, Uppsala, Sweden.
J Biochem Biophys Methods. 2000 Jul 10;44(1-2):1-30. doi: 10.1016/s0165-022x(00)00066-x.
At present, centrifugation is the most common method for separation and isolation of cells and subcellular particles. The technique can be used for a wide range of applications. During latter years it has become obvious what a powerful method density gradient centrifugation is, especially when used in conjunction with sensitive assays or clinical treatments. The most active areas for use of density gradient centrifugation include purification for in vitro fertilization of sperm of both human and bovine origin, isolation of cells for cell therapy of patients receiving chemo- and radiation therapy and basic research both on cellular and subcellular levels. These treatments and investigations require homogeneous populations of cells and cell organelles, which are undamaged after the separation procedure. Percoll, once introduced to reduce convection during centrifugation, has proved to be the density gradient medium of choice since it fulfills almost all criteria of an ideal density gradient medium. Recently good results have also been obtained after silanization of colloidal silica particles, e.g. BactXtractor. The latter medium has proved to be useful in recovery of microorganisms from food samples free of inhibitors to the Polymer Chain Reaction (PCR). The separation procedures described for Percoll in this review seem to be applicable to any cells or organelles in suspension for which differences in size or bouyant density exist. Furthermore, since Percoll media are inert, they are well suited for the separation of fragile elements like enveloped viruses.
目前,离心是分离和纯化细胞及亚细胞颗粒最常用的方法。该技术有广泛的应用。近年来,密度梯度离心已成为一种极为强大的方法,特别是当它与灵敏的分析方法或临床治疗联合使用时。密度梯度离心最活跃的应用领域包括纯化人源和牛源用于体外受精的精子、为接受化疗和放疗的患者进行细胞治疗而分离细胞以及细胞和亚细胞水平的基础研究。这些治疗和研究需要均一的细胞和细胞器群体,且在分离过程后不被破坏。Percoll 自引入用于减少离心过程中的对流以来,已被证明是理想的密度梯度介质,因为它几乎满足了理想密度梯度介质的所有标准。最近,胶体二氧化硅颗粒(如 BactXtractor)硅烷化后也取得了良好的效果。后一种介质已被证明可用于从不含聚合酶链反应(PCR)抑制剂的食品样本中回收微生物。本综述中描述的 Percoll 分离程序似乎适用于任何存在大小或浮力密度差异的悬浮细胞或细胞器。此外,由于 Percoll 介质是惰性的,它们非常适合分离像包膜病毒这样的脆弱成分。