Separation of ovine oligodendrocytes into two distinct bands on a linear sucrose gradient.

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.