原代神经细胞培养方法 Neuron Cell Culture.docVIP

1. Preparation of coverslips 1.1- Mass culture Our standard mass cultures are plated on astrocytes. Those, in turn, are plated on glass coverslips pre-coated with poly-D-lysine and laminin. Materials: . #1 coverslips . coverslip racks in a water-tight container (we made ours) . poly-D-lysine (PDL) stock solution (1mg/ml in dd water) . laminin stock solution (20 (g/ml in Hank’s BSS) . 35 mm plastic culture dishes . culture hood equipped with UV lamp . sterile dd water Procedure: . Place the coverslips in the racks and leave them in the culture hood under UV light for 2 hrs. . Coat the coverslips with 12.5 (g/ml PDL (5ml PDL in 400ml sterile dd water) for 2hrs. in the culture hood. . Wash the coverslips with sterile dd water five times, in the culture hood. . Place the coverslips in the sterile 35mm dishes . Add 0.4ml laminin on top of the coverslips. Wait for 45’, then aspirate the excess solution 1.2- Agarose-collagen microislands This protocol is based on protocols by Segal and Furshpan. Although the following “macro-island” approach has allowed for greater neuronal survival, while still providing a high probability of connection between DRG and dorsal horn (DH) autapses or DH-DH connections can only be obtained with high probability in the conventional microislands. Materials: . #1 coverslips coated with PDL as above . type II agarose . Vitrogen 100 collagen, ~3 mg/ml . 35 mm plastic culture dishes . culture hood equipped with UV lamp . sterile dd water . atomizer Procedure: . Place coverslips in 35 mm dishes . Melt agarose in dd water at 0.2%, and place a drop on the top surface of each coverslip. The height of each drop is diminished as much as possible by removing excess solution with a pipette before the agarose gels. . Allow coated coverslips to air dry overnight in the culture hood at room temperature to form a thin film. For adequate drying, the dishes must be uncovered. . Spray the collagen onto the coverslips with the atomizer. We use a glass per