Selective cultivation of N-cadherin expressing cells from the optic tectum of the chick.
Dissociated primary cell cultures of the nervous system are usually composed of many different cell types, which makes it difficult to investigate a specific cell type and to describe its development in vitro without direct or indirect influence of other cell types. Although various methods have been published to specifically separate either neurons or glial cells, there is still a need for simple protocols to isolate distinct neuronal subpopulations. Here we describe a method to purify specific neuronal subtypes from the chick embryonic midbrain. Embryonic (E10) optic tecta were dissociated and a cell suspension was produced. Cells were separated by magnetic cell sorting (MACS) based on their specific expression of somatic N-cadherin. After cultivation on poly-D-lysine coated dishes in serum-free culture medium supplemented with B27, cells were fixed and analyzed with immuncytochemistry. Enriched primary cultures contained about 70% of N-cadherin positive cells compared to 46% before sorting. 7 days after cultivation, N-cadherin expression and its co-localization with synapses was demonstrated.
Moenig
Benedikt
B
Institut für Biologie II, RWTH Aachen, Germany. moenig@bio2.rwth-aachen.de
Luksch
Harald
H
D000818:Animals; D015820:Cadherins / Q000096:biosynthesis* / Q000276:immunology; D002469:Cell Separation; D002642:Chick Embryo; D018920:Coculture Techniques; D004594:Electrophysiology; D000939:Epitopes / Q000276:immunology; D007150:Immunohistochemistry; D009474:Neurons / Q000378:metabolism; D013477:Superior Colliculi / Q000166:cytology / Q000378:metabolism*
D015820:Cadherins; D000939:Epitopes