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Neurite outgrowth assay: 1x105 neurons were plated into XONA-microfluidic chambers (standard neuron device, SND450) that had been placed on PDL coated coverslips, according to the manufacturers’ instructions. After 4h the plated cells were infected with the respective viruses (scr. shRNA-EGFP control, or NrCAM shRNA-EGFP, 1:1000). Cells were kept until DIV3 at 37°C and 5% CO2, and then the first photomicrographs of the fluorescent neurons were taken with a Leica DM6000 inverted microscope. The images covered the whole channel area in the middle of the respective chambers. Afterwards, the neurons were treated with GI254023x (5 µM), or vehicle and kept at 37°C and 5% CO2 for 24h. At DIV4, a second set of photomicrographs of the same areas were taken and analyzed for length differences of single neurites (length in mm at 24h-0h) with Leica LASX software. Only neurites that had already entered and not yet left the channels on the other side at the timepoint 0h were used for the calculation. When neurites were separating after leaving the main channel the longest process was quantified.
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