Recombinant human β ngf

Isolated pDCs (5 × 10⁴ cells/96-well cavity) were seeded in 200 µl of RPMI 1640 medium (Thermo Fisher Scientific) supplemented with 100 IU ml⁻¹ penicillin, 100 µg ml⁻¹ streptomycin, 2 mM l-glutamine, 10% (vol/vol) human serum and recombinant human (rh) IL-3 [10 ng ml⁻¹; R&D Systems]. To determine the levels of secreted IFNα, pDCs were cultured for 12–14 h in the presence of ODN 2216 (CpG A; 0.26 µM; Thermo Fisher Scientific). To block p75NTR activity, the inhibitory peptide TAT-Pep5 (100 nM, Merck) was added. Recombinant human β-NGF (R&D Systems) was added at the indicated concentrations. The IFNα levels in supernatants were determined using an ELISA (eBioscience). To determine the levels of secreted IL-6, pDCs were stimulated with an antibody specific to human FcεRlα (250 ng ml⁻¹; eBioscience) alone or in the presence of the TAT-Pep5 inhibitor. Recombinant human β-NGF was added at the indicated concentrations. After 14 h, the supernatant was collected to measure IL-6 levels using an ELISA (eBioscience).

Neuroscreen-1 PC12 cells (Thermo Scientific) were plated at 75,000 cells per well on PDL-coated Greiner 96-well tissue culture plates in Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (Life Technologies). Cells were stimulated by the addition of recombinant human β-NGF (R&D Systems, 5 ng/mL final assay concentration), which had been pre-mixed with anti-NGF or isotype control antibodies. 15 min after addition of the NGF, cell supernatants were rapidly removed and cells lysed in 50 μL of lysis buffer from Phospho-ERK Cellular HTRF Assay Kit (Cisbio, Codolet, France). pERK HTRF assay was run according to manufacturer’s instructions.