Culture and identification of embryonic rat spinal motoneurons in vitro / 中华神经科杂志

ABSTRACT

Objective To explore the dissociation, purity and the culture method of spinal motoneurons in vitro and to find out an effective way to identify spinal motoneurons.Methods Spinal motoneurons were isolated from spinal cord of embryonic rat. Spinal tissues were digested and then the motoneurons through 6.8% metrizamide density gradient centrifugation, were obtained by collecting the top layer of metrizamide cushion. After the glia adhered to the plate wall, the enriched motoneurons were collected and plated in 24-well plates at a density of 4?10~5/ml. Cytosine arabinoside was added to the culture system after 24 hours in order to inhibit the outgrowth of glia. L-15 serum medium was used for the first 48 hours and then changed by serum-free medium. Later, neurons were cultured with 50% exchanged medium every two days. Spinal motoneurons were identified by immunostaining with polyclonal antibody to choline acetyltransferase (ChAT) according to ABC method. Laser scanning confocal microscope was used to observe its structure.Results Spinal motoneurons were immunoreactive to the antibody against ChAT. The present experiment revealed that ChAT-positive cells more than 85% might live about seven to nine days.Conclusions This study suggests a method of spinal motoneuron culture successful. Spinal motoneurons could be specially labeled with ChAT antibody.