Poly d lysine hydrobromide

DRG were collected from 2–4‐week‐old Sprague‐Dawley (SD) rats. Bilateral DRG were cultured in vitro in a 35 mm culture dish and studied under a microscope. Next, neuronal fibers connected to the DRG were removed and sensory neurons were isolated from the DRG by sequential digestion using papain and type I collagenase (1 mg mL⁻¹) for 3 h, as reported previously.^[66] Thereafter, 5000 cells mL⁻¹ of these isolated cells were cultured in a Neurobasal medium (A358290, Thermo Fisher Scientific) containing 2% B27 (A3582801, Thermo Fisher Scientific), 0.3% L‐glutamine (25030081, Thermo Fisher Scientific), 100 ng mL⁻¹ NGF (N2513, Sigma‐Aldrich), 1% PSN, 10⁻⁵ M fluorodeoxyuridine (F0503, Sigma‐Aldrich), and 10⁻⁵ M uridine (U3750, Sigma‐Aldrich). These cells were cultured in a six‐well plate coated with poly D‐lysine hydrobromide (50 ng mL⁻¹; Life Technologies). The following day, the medium was replaced with one containing different treatment reagents; subsequently, the medium was refreshed every two days. SiRNA transfection (siRNA ID#: s155625, Thermo Fisher Scientific) (Sequence (5′→3′): Sense: CCGUGGGUGUCUAACUUCAtt; Antisense: UGAAGUUAGACACCCACGGct.) was applied in vitro to knockdown the expression of Sema5b.

Tissue culture media, N2 supplement, antibiotics and fetal calf serum (FCS) and NP40 cell lysis buffer (10×) were obtained from Life Technologies (San Giuliano Milanese, Italy); poly-D-lysine hydrobromide (mol wt 70,000–150,000), poly-L-lysine hydrobromide (mol wt 70,000–150,000), cytosine β-D-arabinoside, 3,3′,5-triiodo-L-thyronine, L-thyroxine, rapamycin, luteolin, papain, DNase I (bovine pancreas), trypsin inhibitor, protease inhibitor cocktail, Pefabloc® SC (100 mM), Pluronic F68 and all other biochemicals were purchased from Sigma-Aldrich (Milan, Italy) unless noted otherwise; Falcon tissue culture plasticware was purchased from BD Biosciences (SACCO srl, Cadorago (CO), Italy). Sterilin petri plastic dishes (10 cm Ø) were from Sarstedt (Verona, Italy). Co-ultramicronized PEA/luteolin (10:1 mass ratio) and ultramicronized PEA were kindly provided by Epitech S.p.A., Saccolongo (PD), Italy.