Separation of ovine oligodendrocytes into two distinct bands on a linear sucrose gradient
Separation of ovine oligodendrocytes into two distinct bands on a linear sucrose gradient
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在线性蔗糖梯度上将绵羊少突胶质细胞分离成两个不同的条带
DOI：
10.1016/0165-0270(80)90030-8
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发表时间：
1980
影响因子：
3
通讯作者：
P. Polak
中科院分区：
文献类型：
--
作者：
S. Szuchet;B. Arnason;P. Polak
本文介绍了一种分离少突胶质细胞的新方法。该方法被开发用于分离完整的活细胞和破碎的少突胶质细胞亚群。将切碎的绵羊白色物质（WM）在0.1%胰蛋白酶中于37 ℃孵育3.6 min/g 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（孔径为350 μm至30 μm）进一步破碎组织，并将0.9 M蔗糖中的粗混悬液以2100 rpm（850 g）离心10 min。在此步骤中，髓磷脂漂浮至试管顶部，同时细胞形成沉淀。将沉淀物重悬于3-4 ml 0.9 M蔗糖中，并应用于线性蔗糖梯度（1.0-1.2 M），然后以1200 rpm（277 g）离心40 min。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。这些条带中的细胞大小存在微小差异。通过这种方法分离的少突胶质细胞保持活力和分化数月，证明了他们的能力，将标记的前体到半乳糖苷和硫苷脂，并合成髓鞘碱性蛋白。
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°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 μm down to 30 μm 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 M surcrose and applied to a linear sucrose gradient (1.0–1.2 M), which is then centrifuged at 1200 rpm (277g) 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.