Quantitative Analysis of Neuronal Differentiation

SH-SY5Y cells were seeded 24 h prior to differentiation in 24-well plate to a density of 1 × 10³ cells per well. Cells were differentiated in B-27™ Plus neuronal culture system (Life Technologies) supplemented with 20 µM retinoic acid (Merck Life Science), 1% L-Glutamine (Gibco), and 1% penicillin/streptomycin (Gibco). Medium was refreshed every other day. Phase-contrast imaging was done on a Zeiss Axiovert 200 M microscope equipped with a Zeiss AxioCam MR3 camera and 20× phase contrast objective. Three images per well were captured at day 8 of differentiation. To extract the total area covered with neurites and soma in each image, we used a custom-developed ImageJ script to automatically segment both neurites and soma, using combinations of simple image operations that can i) remove noise (noise reduction filters), ii) separate fine from coarse structures (rolling ball algorithm or Fast Fourier Transform), iii) separate bright from dark regions (automatic intensity thresholding) and iv) exclude segmented regions based on size or shape (morphological operations). The resulting segmentation masks were used to calculate the ratio of skeletonized neurites per cell body area. In addition, we manually traced individual neurite structures. To this end, images were converted to 8-bit and analyzed with NeuronJ plugin in ImageJ, a commonly used tool for semiautomatic tracings and measurements of neurites^{67 (link)}. Any projection from SH-SY5Y cell body was considered a “primary neurite”, whereas projections branching from primary neurites were considered a “secondary neurite”. Three wells per genotype and three images per well were analyzed, and data from two experiments (repetition) was pooled, resulting in over 600 tracings in total. The overall distribution of primary and secondary neurites lengths was plotted. For each image, the fraction of secondary neurites (over the total) was also calculated. One-Way Anova was used for statistical analysis.