Bsa gradient

Lumbar dorsal root ganglia (DRG) was harvested from adult mice (age >18 weeks) as previously published (2 (link), 29 (link)). After removal of the connective tissue from the ganglia, they were incubated two times in Liberase (Roche, 9 mg 100 ml^−l DMEM) for 30 min. After washing with phosphate-buffered saline (PBS), the tissue was incubated with Trypsin-EDTA (0.05%, Gibco, Life Technologies) for 15 min and subsequently washed with TNB medium (Biochrom, Merck Millipore) supplemented with L-glutamin (0.2 mM), Penicillin and Streptomycin (200 U ml^−l, Gibco, Life Technologies), and Protein-Lipid Complex (Biochrom, Merck Millipore). The DRGs were dissociated with a fire-polished Pasteur pipette and centrifuged through a 3.5% BSA gradient (Sigma) to eliminate debris and non-neuronal cells. The pelleted sensory neurons were resuspended and were plated on coverslips coated with poly-L-lysine/laminin-1 (Sigma), and cultivated in supplemented TNB containing mNGF 2.5 s (Alomone Labs, 25 ng ml^−l TNB medium) at 37°C and 5% CO2 in a humidified incubator for 16–24 h.

Lumbar DRG containing the cell bodies of primary afferents that project into the hindpaw were harvested from adult male mice (age 8–16 weeks) as previously published
[58 (link), 59 (link)]. After removal of the connective tissue, ganglia were incubated in Liberase Blendzyme 1 (9 mg/100 ml DMEM, Roche) for 2 times 30 min. After washing with PBS (PAA), 1× Trypsin-EDTA (Invitrogen) was added for 15 min. and DRG were washed with TNB™ medium (Biochrom) supplemented with L-glutamin (Invitrogen), penicillin G sodium, streptomycin sulfate (Invitrogen), and Protein-Lipid-Komplex™ (Biochrom). The DRG were dissociated with a fire-polished Pasteur pipette and centrifuged through a 3.5% BSA gradient (Sigma) to eliminate non-neuronal cells. The sensory neurons were resuspended, plated on coverslips coated with poly-L-Lysine/laminin-1 (Sigma), and cultivated in supplemented TNB™ containing mNGF 2.5S (Alomone Labs, 10 μg/100 ml TNB-medium) at 37 °C in 5% CO2 for 24–36 h.

Lumbar (L1–L6) DRG containing the cell bodies of primary afferents that project into the hindpaw were harvested from adult C57BL/6J mice (age 8–12 weeks) as previously published (Camprubí-Robles et al., 2013 (link); Langeslag et al., 2014 (link)). Briefly, ganglia were treated enzymatically with Liberase Blendzyme 1 (9 mg/100 ml DMEM, Roche) for two times 30 min and 1× Trypsin-EDTA (Invitrogen) for 15 min. The DRG were then washed and dissociated mechanically in serum-free TNB^® medium (Biochrom) with a fire-polished Pasteur pipette, and centrifuged through a 3.5% BSA gradient (Sigma) to eliminate non-neuronal cells and debris. The resulting sensory neurons were resuspended, plated on poly-L-lysine/laminin coated coverslips and cultivated in TNB medium supplemented with NGF (25 ng/ml), L-glutamine, penicillin G sodium and streptomycin sulfate (all from Invitrogen) at 37°C in 5% CO₂ for 24 h, unless otherwise indicated.