Dm6000 inverted microscope

1 × 10⁵ neurons were plated into XONA‐microfluidic chambers (standard neuron device, SND450) that had been placed on PDL‐coated coverslips, according to the manufacturer's instructions. After 4 h, 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. Afterward, the neurons were treated with GI254023x (5 μM), or vehicle and kept at 37°C and 5% CO₂ for 24 h. 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 24–0 h) with Leica LASX software. Only neurites that had already entered and not yet left the channels on the other side at the timepoint 0 h were used for the calculation. When neurites were separating after leaving the main channel, the longest process was quantified.

The sorted ILC2 and T-cells were fixed in PBS containing 1% paraformaldehyde, washed with PBS, stored, and protected from light until analysis. Sub-membrane actin and nuclei (DNA) were labeled with Alexa Fluor 488 Phalloidin (Sigma-Aldrich, Munich, Germany) and Hoechst 33258 (Sigma-Aldrich), respectively (32 (link)). The cells were either transferred to optical-bottom 12-well plates (Thermo Fisher Scientific) in PBS for observation by confocal microscopy (32 (link)). The samples were analyzed using a Leica DM6000 inverted microscope connected to a Leica SP5 laser scanning confocal system and Fiji software to determine the cell integrity after sorting.

Cells were fixed with 4% formaldehyde for 15 min and permeabilized with 0.5% Triton X-100 in PBS for 10 min. The cells were blocked with 10% donkey serum in PBS for at least 1 h. Primary antibodies against Nestin (Abcam, ab92391), SOX2 (Abcam, ab59776), SOX1 (R&D Systems, AF3369), PAX6 (BioLegend, PRB-278P), Ki67 (CST, 9449S), Dlx2 (Abcam, ab117546), FOXG1 (Abcam, ab18259), MAP2 (Sigma Aldrich M9942), TBR1 (Abcam ab31940), BRN2 (Cell Signaling 12,137), NeuN (Abcam ab104225), SOX9 (R&D Systems AF3075), GLT1 (Novus Biologicals NBP1–20136), and GFAP (Dako Z0334) were diluted in blocking buffer and incubated overnight at 4 °C. Following several washes, the cells were incubated with the appropriate donkey anti-rabbit IgG, anti-mouse IgG, or anti-goat conjugated with Alexa Fluor 488, Alexa Fluor 555, or Alexa Fluor 647 secondary antibodies (Life Technologies) for 1 h at room temperature. Cells were counterstained with DAPI and visualized with the Leica DM6000 inverted microscope. Images were acquired using the Q-Imaging Retiga Xi Firewire High-Speed, 12-bit cooled CCD camera and Volocity software. Staining was quantified using ImageJ software and represented as a portion of total nuclei. On average, 2.5 × 10³ cells were quantified from each experiment. Results are shown with the standard deviation of the mean of three independent experiments.