Hippocampal Neuron Transfection Protocol

Hippocampal low density cultures were prepared as described previously (Goslin et al., 1998 ). Neurons were plated at an approximate density of 70 cells/mm² and were transfected using a modified calcium phosphate precipitation method (Kohrmann et al., 1999 (link)). Briefly, for transfection of a 6-cm dish, 6 μg of plasmid DNA was mixed with 120 μl of 250 mM CaCl₂ in a polypropylene tube. 120 μl of 2× HBS (274 mM NaCl, 9.5 mM KCl, 15 mM glucose, 42 mM Hepes, 1.4 mM Na₂HPO₄, pH 7.10–7.15) was then added dropwise to the mixture with aeration. This mixture was added immediately to a 6-cm dish of the neurons with 4 ml of 24-h glia-conditioned medium. When complex formation was observed (typically 30–60 min), the cells were washed twice with HBS (135 mM NaCl, 4 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 10 mM glucose, 20 mM Hepes, pH 7.35), and then glia-conditioned medium with 0.5 mM kynurenic acid was added. Using this method, the transfection efficiency for the hippocampal neurons ranged from 10 to 30%.