Neural Crest Cell Migration and Proliferation

Digital images of the outgrowth cultures at 24–48 h were acquired by video microscopy. Then NIH Image software was used to measure the area of the outgrowth. This was obtained by taking the area of the entire explant and subtracting from it the area of the dense central neural tube mass. Since the neural tube fragment is attached to the substratum predominantly via its edges where neural crest cells are emerging, we controlled for differences in migration area resulting from random variations in the shape of the explant by dividing the outgrowth area (mm²) by the perimeter (mm) of the explant. This normalized number (in mm) is referred to as the migration index and provides an estimate of the migration rate of the neural crest cells. For monitoring cell proliferation, 48-h explant cultures were incubated with bromodeoxyuridine (BrdU) (10 μM/ml) for 2 h, fixed, and processed for immunodetection using an anti-BrdU antibody and a streptavidin peroxidase detection system (Calbiochem, San Diego, CA) used in conjunction with the True Blue™ peroxidase substrate (Kierkegaard and Perry Laboratories, Gaithersburg, MD). Labeled cells were counted with the aid of NIH Image. To determine the total cell number in the outgrowth after BrdU immunostaining, the cultures were further stained with hematoxylin. This permitted a total cell count to be obtained using NIH Image. The proliferation rate was then calculated as the percent of BrdU-labeled cells in the outgrowth.