The Role Of Calcium And Calmodulin Dependent Kinases For Neurons Against Hypoxia And Its Intracellular Mechanism

Part I Primary culture and indentification of embryonic cerebral cortical neurons of a rat and establishment of hypoxic injured neuron cell-model in vitroObjective: To culture and obtain the pure embryonic cortical neurons of a rat in vitro and identify the ratio of neurons among the whole primary cultured neuron cells. We also established a hypoxic injured cell-model of cerebral cortical neurons of a rat embryo in vitro using a neural culture system and evaluated this system with immunofluorescence technical methods.Methods:1. Primary embryonic cortical neurons of a rat were dissociated and digested by 0.05% Trypsin and 10IU/ml DNase I and cultured in Neurobasal medium supplement 2% B27,5% fetal serum in vitro. 1% Glutamax was added to Neurobasal Medium to replace glutamine, which could support the plating and culturing of the neurons. Then 10umol/L cytosine arabinoside was added to inhibit glial growth after plating 48 hours. 2. The purity of the cultured embryonic cortical neurons was assessed by immunofluorescence technique which stained neurons with anti-tubulinβ-III monoclonal antibodies and stained astroglials with anti-GFAP polyclonal antibodies. 3.