parental PC12 cells and/or TrkB-PC12 were harvested from T-75 flasks, centrifuged and subjected to the needle aspiration procedure described above. Cells were seeded on 100 × 20 mm Corning BioCoat collagen I-coated dishes (Fisher Sci #356450) in complete growth medium at a density of 1 × 106 cells/ml (10 ml per dish) and grown for one day at 37°C in 5% CO2. Before stimulation with ligands, cells were starved overnight in RPMI-1640 medium containing L-glutamine, HEPES, sodium pyruvate and penicillin-streptomycin. PC12 or TrkB-PC12 cells transiently transfected with siRNA p75NTR or control siRNA for 72 hours were left unstimulated or were stimulated with 50 ng/ml BDNF (R&D Systems #248-BD reconstituted in PBS with 1 mg/ml BSA) or 50 ng/ml rat NGF from R&D Systems (#556-NG-100) for the time intervals shown at 37°C in humidified 5% CO2. Cells were lysed using ice-cold Cell Lysis Buffer (CST #9803) supplemented with Phosphatase Inhibitor Cocktail Tablets PhosSTOP (Roche #04906837001) and Protease Inhibitor Cocktail Tablets Complete (Roche #11697498001). Cells were scraped from the surface, harvested, vortexed, incubated for 30 min on ice, and then centrifuged at 10,000 × g for 10 min at 4°C. Aliquots of supernatant were dissolved in NuPAGE LDS sample buffer (ThermoFisher Sci #NP0007) supplemented with 50 mM DTT and heated for 7 min at 75°C.
Rat ngf
Manufactured by R&D Systems
Sourced in France
Rat Nerve Growth Factor (NGF) is a recombinant protein that promotes the growth and survival of sympathetic and sensory neurons. It is a member of the neurotrophin family of growth factors.
Automatically generated - may contain errors
Lab products found in correlation
Phosphatase inhibitor cocktail tablets phosstop, by Roche (1 mentions)
Protease inhibitor cocktail tablets complete, by Roche (1 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Streptavidin cryptate, by PerkinElmer (1 mentions)
Brain derived neurotrophic factor (bdnf), by R&D Systems (1 mentions)
3 protocols using rat ngf
1
Monitoring NGF/BDNF Signaling in PC12 Cells
2
Differentiation of PC12 Cells with NGF
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The PC12 cell line was a generous gift from Dr Frédérique René (Faculté de medicine, Strasbourg, France). All products are from Gibco, except rat NGF from R&D systems, France. Cells were routinely cultured in RPMI 1640 medium complemented with 10% horse serum, 5% decomplemented fetal bovine serum, 2 mM L-glutamine, 100 U.mL-1 penicillin and 100 µg.mL-1 streptomycin and subcultured once a week at 25,000 cells/cm2 after trypsinisation, with medium changes every 2–3 days. For differentiation assays, cells were seeded at 10,000 cells/cm2 in differentiation medium composed of RPMI 1640 complemented with 1% horse serum, 2mM L-glutamine, 100 U.mL−1 penicillin and 100 µg.mL−1 streptomycin. The medium was renewed every 2–3 days. Cells were incubated for 7 to 11 days at 37°C in an atmosphere of 95% air and 5% CO2. For positive controls, 50 ng.mL−1 of rat NGF was added in differentiation medium. Statistical analysis was performed using InStat software (Software Inc., GraphPad, San Diego, CA). Values were expressed as mean ± standard deviation (mean ± SD). Neurites per cell and neurites lenght from each group were compared using nonparametric ANOVA (Kruskal-Wallis Test and Tukey post hos test). A p-value less than 0.05 was considered to be significant.
3
Characterizing MEDI1912 IgG Specificity
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The specificity of MEDI1912 IgG was established using a Homogeneous Time Resolved Fluorescence (HTRF®) epitope competition assay. The assay determines relative cross reactivity by measuring inhibition of biotinylated NGF (in house HEK-EBNA derived), binding to MEDI1912 IgG. Binding of MEDI1912 IgG to biotinylated NGF is detected by FRET between an XL6650 labelled anti-human-Fc antibody (CisBio) that binds MEDI1912 IgG and streptavidin cryptate (CisBio) which binds biotinylated NGF. A panel of NGF related proteins (NT-3; NT-4; BDNF; murine NGF; and rat NGF (R&D Systems)) were titrated in the assay and any proteins which bound to MEDI1912 IgG caused a dose dependent decrease in FRET.
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