Isolation of fetal mouse motor neurons on discontinuous percoll density gradients |
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Authors: | Michael J. Strong Ralph M. Garruto |
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Affiliation: | (1) Laboratory of Central Nervous System Studies, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bldg. 36, Rm. 5B-21, 20892 Bethesda, Maryland |
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Abstract: | Summary The spinal cords of fetal NIH∶CR mice, gestational age Day 12 to 14, were dissected free of meninges and dorsal root ganglia, chemically dissociated, and layered onto discontinuous Percoll gradients at densities 1.040, 1.050, and 1.060 g/ml. After centrifugation (800 Xg for 15 min at 4° C), three morphologically, biochemically, and immunohistologically distinct cell populations were collected from the gradient interfaces. The first interface, located at a density of 1.040 g/ml, was choline acetyltransferase enriched (0.86±0.08) compared to the second and third fractions (0.42±0.01 and 0 pmol acetylcholine synthesized/μg protein, respectively). When simultaneously cultured with fetal mouse cardiac muscle on a gelatin-polylysine-laminin substrate in serum-free medium, these cells developed the characteristics of motor neurons. Dr. Strong is supported in part by a Research Fellowship from the Medical Research Council of Canada. |
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Keywords: | motor neurons Percoll density gradient choline acetyltransferase |
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