25-Hydroxy Vitamin D Exhibits NGF-like Activity in PC12 Cells

Abstract

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.