Materials and Methods
ProductsNGFβ from rats was purchased from (Sigma, USA). 25OHD was purchased from Immundiagnostik AG (Bensheim, Germany).
Cell culture and differentiationThe rat adrenal pheochromocytoma-derived cell line PC12 was obtained from RIKEN Bio Resource Research Center (Ibaraki, Japan) and maintained in Dulbecco's modified Eagle's medium (Thermo Fishier Scientific, Japan), supplemented with 10% deactivated horse serum, 10% deactivated bovine serum, 100 unit/mL penicillin and 100 µg/mL streptomycin sulfate, at 5% CO 2 , 37 °C. PC12 cells were seeded at a density of 1.5 x 10 5 cells. After 24h of incubation, the PC12 cells were treated with 10ng/mL NGF with or without 6 nmol/L of 25OHD for 9 days.
Measurement of neurite elongationPhase-contrast micrographs were recorded on a personal computer using a charge coupled device camera (Ikegami, Japan), followed by analysis using software (Miotic Images Plus 2.1S; Shimadzu, Japan). Cells with protrusions longer than the cell body diameter were counted as cell with neurites. PC12 cells were treated with NGF for 9 days for the proper tracing of neurites belonging to a specific cell body.