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Efficient DNA transfection of primary CNS neurons using TransMessenger ,,, Transfection Reagent

on poly-D-lysine-coated 96-well plates at a density of 120,000 cells/well in a volume of 200 l/well.

Cells were transfected in a 96-well format on the seventh day after plating. The indicated amounts of DNA (0.1-0.5 g/well of a GFP- or -gal-encoding plasmid) were condensed for 5 minutes with Enhancer R and Buffer EC-R in a total volume of 30 l. The ratio of DNA to Enhancer R was 1:4. TransMessenger Reagent (1-4 l) was added in a total volume of 20 l Buffer EC-R and complex formation proceeded for 10 minutes. 100 l Neurobasal/B27 medium was added to the transfection complexes, medium was removed from the cells, and the complexes were transferred to the cells. After 6 hours of incubation, the complexes were removed and fresh medium was added.

For immunofluorescence, cells were fixed using 4% paraformaldehyde 24 hours after transfection. Immunofluorescence was performed according to standard procedures using primary antibodies directed against MAP2 (a neuron-specific marker protein) and GFAP (an astrocyte-specific marker protein) and Cy3-labeled secondary antibodies.

-galactosidase activity was measured 48 hours after transfection. Cells were lysed and enzyme activity was quantified by a colorimetric assay using ONPG and a -galactosidase standard curve.

Results and discussion

To optimize the transfection protocol, transfections were performed using different amounts of plasmid DNA and TransMessenger Reagent. In each well of a 96-well plate, 0.1, 0.3, or 0.5 g plasmid DNA were used, in combination with 1-4 l of TransMessenger Reagent. Six replicates were set up for each combination.

Transfection efficiencies were quantified by measurin
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