2.7. Neurite Outgrowth Assay and Quantification

The NSCs were plated on 12-well plates coated with poly-L-ornithine and laminin at a density of 3 x 10⁴ cells per well in StemPro NSC SFM medium and incubated for 48 h at 37°C in 5% CO₂. The NSCs were treated with varying concentrations of L. rhinocerus HA or ME extracts (1 - 100 μg/ml) in Neurobasal medium supplemented with 2 mM GlutaMAX-I supplement and 1% (v/v) Penicillin-Streptomycin solution to determine the optimal concentration that induced maximal neurite outgrowth. In addition, NSCs were treated with DEX (1 - 100 μM) to study the GC-induced effect on neurite outgrowth. Cells cultured in medium without L. rhinocerus sclerotial extract or DEX served as the negative control, and cells grown in medium with 2% (v/v) B-27 supplement were the positive control. Cells were incubated for 72 h at 37°C in 5% CO₂ and assessed for neurite outgrowth.

For the quantification of neurite outgrowth, five random fields from each well were photographed and examined for cells with neurite outgrowth, i.e., cells with axon-like extensions that were at least twice the length of the cell body diameter by blind analysis [14]. The total number of cells with neurites and total number of viable cells per well were counted in the ImageJ software. The percentage of neurite-bearing cells was obtained by calculating the ratio of neurite-bearing cells to the total viable cell number in each well.

To further evaluate the neurite outgrowth and extension of NSCs, the cells were plated in 8-well chamber slides and treated with the optimal concentration of L. rhinocerus HA and ME extracts as well as DEX (1 – 100 μM) for 72 h. The NSCs were then stained with mouse anti-TUJ-1 primary antibody and goat anti-mouse IgG-FITC secondary antibody following the aforementioned protocol for immunostaining. The slide was mounted and observed under a Nikon Eclipse Ti-S inverted fluorescence microscope and images were captured using the Nikon NIS-Elements microscope imaging software.