Incucyte neurotrack analysis software module

Mouse primary neurons in 24-well plates were infected at DIV 2 with rAAV/Lnc473 or rAAV/EGFP. At DIV 7, transfection with a pSYN1-mScarlet plasmid was performed (0.5 μg DNA/well, 1:2 DNA (μg) to LF (μl) ratio). Plates were transferred into an Incucyte (Sartorius) live cell imaging incubator and phase contrast as well as EGFP and mScarlet fluorescence images were obtained at 4 h intervals during DIV 9 to 12.5. mScarlet expression and the Incucyte Neurotrack Analysis Software Module (Sartorius; Incucyte-neurotrack-analysis-software">https://www.sartorius.com/en/products/live-cell-imaging-analysis/live-cell-analysis-software/Incucyte-neurotrack-analysis-software) with Top-Hat cell body cluster segmentation, 5 μm minimum cell width cleanup, 100 μm² minimum cell body area filter, and neurite coarse and fine sensitivity set at 9 and 0.7, respectively, were used to define cell somas and generate neurite traces of transfected neurons. In each well total neurite length per cell was quantified.

To analyze the neurite complexity, the Incucyte^®® S3 live-cell imaging system (Sartorius, Minisart, Göttingen, Germany) was used. A total of 4000 PC12 cells stably expressing mCherry were seeded on collagen coated Costar^®® 24-well Clear TC-treated Multiple Well Plates (Corning, Corning, NY, USA). Differentiation was induced with RPMI 1640 medium with 1% fetal horse serum and 0.5% fetal bovine serum containing 50 µg/mL NGF. This medium was refreshed every 48 h until full differentiation at day 10. Plates were scanned using the Incucyte system using a 20× objective and 36 images were taken per well. Assessment of neurite length and branch points was performed using the Incucyte^® Neurotrack analysis software module (Sartorius, Minisart, Göttingen, Germany). Average neurite length and branch points per cell were calculated by dividing total neurite length or branch points per well by the cell confluence expressed in mm².