Poly l lysine laminin 1

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 were harvested from adult mice as previously published [15 (link), 16 (link)]. After removal of the connective tissue from the ganglia, they were incubated twice in Liberase (90 mg·L⁻¹ DMEM) for 30 min. After washing with PBS, the tissue was incubated with 0.05% Trypsin‐EDTA for 15 min and subsequently washed with TNB medium (Biochrom, Merck Millipore, Berlington, MA, USA) supplemented with l‐glutamin (0.2 mm), penicillin and streptomycin (200 U·mL⁻¹), and protein–lipid complex (Biochrom, Merck Millipore). The DRG were dissociated with a fire‐polished Pasteur pipette and centrifuged through a 3.5% BSA gradient to eliminate debris and non‐neuronal cells. The pelleted sensory neurons were resuspended, plated on coverslips coated with poly‐l‐lysine/laminin‐1 (Sigma‐Aldrich), and cultivated in supplemented TNB containing mNGF 2.5 s (25 ng·mL⁻¹) at 37 °C and 5% CO₂ in a humidified incubator.

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.