Fig. 2: Morphological analysis revealed complex neuronal networks and high expression of NeuN in SH-SY5Y cells cultured in 3D DMAP2 Mix at 10 DIV and/or 40 DIV.
A SH-SY5Y cells were plated and differentiated in 3D DMAP2 Mix condition as described in the Materials and Methods section. Cells were fixed, permeabilized, and immunostained with either the anti-β-tubulin (orange) antibody and Hoechst (blue) to detect neurite networks and nuclei, respectively. A total of 10 z-stacks images for each condition were taken. Maximum projection and large images (6 fields) are shown. Scale bar: 200 µm. B Schematic representation of some parameters taken into account during neuronal arborization analysis. In particular, the main parameters measured were: the number of extremities (C), the number of roots (D), the number of segments (E), the number of nodes type I (F), and the number of nodes type II (G). H The panel illustrates the NeuN (orange) and Hoechst (blue) merged staining at 10 DIV; the overlay is represented in pink. A total of 10 z-stacks images for each condition were taken. Maximum projection and large images (4 fields) are shown on the left. Scale bar: 100 µm. One representative field is shown on the right. Scale bar: 50 µm. I Quantification of NeuN positive cells (histogram); 95 positive neurons out of 99 neurons analyzed from 12 different fields. The totality of fluorescence images was captured using Operetta CLSâ„¢ equipped with a 63Ă— immersion objective. Mann-Whitney test was used to determine the significance of differences among the conditions analyzed. Significance was set as *p < 0.5, **p < 0.01, ***p < 0.001, ****p < 0.0001.
