Isolation of fetal mouse motor neurons on discontinuous Percoll density gradients

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 degrees 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/microgram 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.