Nogo a peptide

PC-12 cells were examined for proliferation through the active incorporation of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) dye (Invitrogen). They were seeded at a cell density of 2000 per well into a 96-well plate and grown overnight prior to receiving treatment. On day 2, cells were treated with 100 ng/mL NGF (R&D Systems, Minneapolis, MN, USA) for 2 h followed by incubation with a recombinant Nogo-A peptide (corresponding to aminoacids 566–748 of the human protein, thus expected to block the 11c7 antibody; R&D Systems) using 1 µg/mL. The cells were then treated with 1, 3, and 12 µg/mL of Rat Nogo-A monoclonal antibody 11c7. PC-12 cells were incubated for 6 days for to monitor axonal growth and neurite maturity. At pre-determined times, cells were treated with 0.5 mg/mL of MTT in complete media for 2 h. The media was then completely removed and replaced with 100 µL of dimethyl sulfoxide (DMSO) for 30 min and the plate was then measured on a Synergy H4 plate reader at optical density (OD) of 570 nm.

PC-12 cells were seeded on collagen-type IV-coated 24-well plates (BD-Biocoat) at a cell density of 4000 cells/cm² and grown overnight prior to receiving treatment. On day 2, cells were treated with 100 ng/mL NGF (R&D Systems) for 2 h followed by incubation with a recombinant Nogo-A peptide (R&D Systems) using 25, 100, and 1000 ng/mL. The cells were then treated with 1, 3, and 12 µg/mL of Rat Nogo-A monoclonal antibody 11c7. PC-12 cells were incubated for 6 days for to monitor axonal growth and neurite maturity.