VSC4.1 motor neurons were maintained in DMEM medium containing 15 mM HEPES, pyridoxine NaHCO3 (Sigma, St. Louis, Missouri, USA), 2% Sato’s components, 1% penicillin and streptomycin (Beyotime, Shanghai, China) as well as 15% heat-inactivated FBS (Hyclone, Logan, UT, USA) in poly-L-ornithine coated 7.5 cm2 flasks. Cells were grown at 37 °C in the incubator with 5% CO2 and full humidity.
Cells were redistributed with DMEM containing 2% FBS at density of 106 cells in 75-cm2 flasks and 2.5–5 × 104 cells/well in 6-well plates for 24 hours, respectively. Prepared Cells were treated with HD (25 mM) or saline for 24 h and then were treated with BMSC-CM (15%, v/v) in the presence or absence of some pathway inhibitors or activators, containing PIK III (20 μM), Rap (20 μM), anti NGF antibody (10 μM), anti IgG antibody (10 μM), NGF (100 μM) and K252a (20 μM), for another 24 h. Cells cultured in DMEM (containing 2% FBS) with equal amount of saline were used as the negative control. Schematic diagram of experimental design about VSC 4.1 cells was showed in Fig.5B .
Cells were redistributed with DMEM containing 2% FBS at density of 106 cells in 75-cm2 flasks and 2.5–5 × 104 cells/well in 6-well plates for 24 hours, respectively. Prepared Cells were treated with HD (25 mM) or saline for 24 h and then were treated with BMSC-CM (15%, v/v) in the presence or absence of some pathway inhibitors or activators, containing PIK III (20 μM), Rap (20 μM), anti NGF antibody (10 μM), anti IgG antibody (10 μM), NGF (100 μM) and K252a (20 μM), for another 24 h. Cells cultured in DMEM (containing 2% FBS) with equal amount of saline were used as the negative control. Schematic diagram of experimental design about VSC 4.1 cells was showed in Fig.