( ps were transferred (cell-side...)Effectene Transfection Reagent provides efficient gene delivery ,,, to primary neuronal cell cultures

ps were transferred (cell-side down) to dishes containing a confluent layer of collicular glial cells and were cultured for up to 3 weeks. On the second day in vitro, cytosine arabinoside (4 M) was added to inhibit glial cell proliferation. Transfection was performed on day 9 in vitro using various ratios of EGFP plasmid DNA (carrying a CMV promoter and isolated from DH5alpha E. coli using a QIAGEN Plasmid Kit) to Effectene reagent (Figure 1). Transfection was performed as described in the Effectene Transfection Reagent Handbook. During DNAEnhancer Effectene Reagent complex formation, the coverslips containing the neurons were removed from the glial cell culture and placed cell-side up in 350 l fresh, pre-warmed culture medium in the well of a 24-well plate. Culture medium (350 l per well) was added to transfection complexes and mixed by pipetting up and down. Diluted transfection complexes were then added to neuronal cultures and incubated for 4.5 hours. After incubation, the coverslips were returned to the glial cell culture (cell-side down), and cells were cultured for 24 hours before analysis of expression. All transfections were performed in triplicate. Control cells were exposed to Effectene Reagent complexed with plasmid DNA lacking a CMV eukaryotic promoter. Immunofluorescence was performed as previously described (2).

Efficient Transfection Using Effectene Reagent

Figure 1 Dependence of transfection efficiencies on transfection conditions. Transfection efficiencies are expressed as the percentage of neurons that are GFP-positive. Note that transfection efficiency decreased when more than 5 l of Effectene Reagent was added. Highest transfection efficiencies were obtained u
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