Szuchet S, Arnason B G, Polak P E
J Neurosci Methods. 1980 Oct;3(1):7-19. doi: 10.1016/0165-0270(80)90030-8.
A new method for isolation of oligodendrocytes is described. The method was developed to isolate intact, viable cells and to fractionate oligodendrocyte subgroups. Finely minced ovine white matter (WM) is incubated in 0.1% trypsin at 37 degrees C for 3.6 min/g WM. Trypsin inhibitor is added to arrest the action of trypsin. Further disruption of tissue is achieved by passage through a series of screens (350 micrometer down to 30 micrometer pore size) and the crude suspension in 0.9 M sucrose is centrifuged at 2100 rpm (850 g) for 10 min. During this step myelin floats to the top of the tube while the cells form a pellet. The pellet is resuspended in 3-4 ml of 0.9 sucrose and applied to a linear sucrose gradient (1.0-1.2 M), which is then centrifuged at 1200 rpm (277 grams) for 40 min. Oligodendrocytes separate into two distinct bands on this gradient suggesting that two subpopulations have been isolated. There are small differences in size between cells from these bands. Oligodendrocytes isolated by this procedure remain viable and differentiated for months as evidenced by their ability to incorporate labeled precursors into galactocerebrosides and sulfatides and to synthesize myelin basic protein.
本文描述了一种分离少突胶质细胞的新方法。该方法旨在分离完整、有活力的细胞，并对少突胶质细胞亚群进行分级分离。将精细切碎的羊白质（WM）在37℃下于0.1%胰蛋白酶中孵育3.6分钟/克WM。加入胰蛋白酶抑制剂以阻止胰蛋白酶的作用。通过一系列筛网（孔径从350微米降至30微米）进一步破坏组织，将0.9M蔗糖中的粗悬液以2100转/分钟（850克）离心10分钟。在此步骤中，髓磷脂漂浮到管的顶部，而细胞形成沉淀。将沉淀重悬于3 - 4毫升0.9M蔗糖中，并应用于线性蔗糖梯度（1.0 - 1.2M），然后以1200转/分钟（277克）离心40分钟。少突胶质细胞在该梯度上分离成两个不同的条带，表明已分离出两个亚群。这些条带中的细胞在大小上存在微小差异。通过该程序分离的少突胶质细胞在数月内保持活力并分化，这可通过它们将标记前体掺入半乳糖脑苷脂和硫脂以及合成髓鞘碱性蛋白的能力来证明。
