Neurite Stimulation by Sclerotium Extract and NGF

Cells were cultured for 2 to 3 days until 60–70% confluent prior to assay. They were plated into 12-well plates at cell density of 5 × 10⁴ cells per well in complete F-12K medium with concentrations 1 to 500 μg/mL (w/v) of sclerotium aqueous extract. Concentrations of NGF-7S from murine submaxillary gland (Sigma, St. Louis, MO, USA) ranging from 10 ng/mL (w/v) to 100 ng/mL (w/v) were tested to examine the optimum concentration for neurite stimulation activity. The optimum concentration was used as positive control for the following assays. Cells in complete F-12K medium without treatment served as negative control. Freeze-dried aqueous extracts were diluted to various concentration with sterilized distilled water. After the preliminary test, optimum concentration of the sclerotium aqueous extract in combination with NGF ranging from 10 ng/mL (w/v) to 50 ng/mL (w/v) was tested to evaluate synergistic interaction, if any, between sclerotium aqueous extract and NGF. Assay plates were incubated at 37 ± 2°C in a 5% CO₂-humidified incubator. Differentiation activity of cells in terms of neurite outgrowth and branching was observed after 48 hr of incubation at 37 ± 2°C in 5% CO₂-humidified incubator.