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Separation of ovine oligodendrocytes into two distinct bands on a linear sucrose gradient
Abstract:
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.
Insights
This study introduces a novel method to isolate intact, viable oligodendrocytes and their subgroups from ovine white matter. The technique successfully separates two distinct oligodendrocyte subpopulations, crucial for myelin research.
Area of Science:
- Neuroscience
- Cell Biology
- Biochemistry
Background:
- Oligodendrocytes are crucial glial cells responsible for myelin production in the central nervous system.
- Efficient isolation of viable oligodendrocytes and their subpopulations is essential for studying myelin development and disease.
Purpose of the Study:
- To develop and describe a new method for isolating intact, viable oligodendrocytes.
- To enable the fractionation of oligodendrocyte subgroups for further research.
- To ensure the isolated cells remain viable and differentiated for extended periods.
Main Methods:
- Incubation of ovine white matter in trypsin, followed by trypsin inhibitor treatment.
- Mechanical disruption using sequential sieving (350 to 30 micrometers).
- Differential centrifugation to separate myelin from cells, followed by density gradient centrifugation (1.0-1.2 M sucrose) to isolate oligodendrocytes.
Main Results:
- The method yields intact, viable oligodendrocytes.
- Two distinct oligodendrocyte subpopulations are separated based on density gradient centrifugation.
- Isolated oligodendrocytes remain viable and differentiated for months, confirmed by precursor incorporation and myelin basic protein synthesis.
Conclusions:
- A novel, effective method for isolating oligodendrocytes and their subpopulations from ovine white matter has been established.
- The isolated cells are viable and maintain differentiation capabilities, suitable for long-term studies.
- This technique facilitates research into oligodendrocyte biology and myelin-related disorders.