Ham s f 12 media

Primary guinea pig gastric epithelial cells were obtained as previously described [54 (link)]. Briefly, the stomach was isolated from the guinea pig, homogenized and treated with 0.25% trypsin (BIOMED-LUBLIN, Lublin, Poland) for 15 min at room temperature. The cell suspension at a density of 2 × 10⁶ cells/mL was seeded onto a 6-well plate (Becton Dickinson, Franklin Lakes, NY, USA) and cultured for 24 h (5% CO₂, 37 °C) to allow the cells to adhere. The cells were cultured in a mixture of DMEM and Ham’s F-12 media (ratio 1:1; Sigma-Aldrich, Saint Louis, Mi, USA), supplemented with 10% fetal calf serum (FCS), 1% (N-2-hydroxyethylpiperazine-N-2-ethane sulfonicacid) (HEPES), penicillin (100 U/mL), streptomycin (100 µg/mL), amphotericin B (0.025 µg/mL), l-glutamine (2 mM/mL)epidermal growth factor (Sigma-Aldrich, Saint Louis, MI, USA) 0.01 µg/mL and 0.005% dexamethasone. The guinea pig fibroblasts (CRL-1405) were obtained from the American Type Culture Collection (ATCC, Rockville, Manassas, VA, USA) The cells were routinely grown as a monolayer in complete RPMI-1640 medium cRPMI(Sigma St. Louis, MI, United States) at 37 °C in a humidified atmosphere containing 5% CO₂.Every 2–3 days, the medium was changed and the cells were passaged at 80–90% confluence.

DRG neurons were isolated from adult male Sprague Dawley (300-350 g) rats, dissociated and cultured using previously described methods [33 (link)]. Briefly, neurons were cultured in glucose-free Hams F12 media supplemented with Bottenstein’s N2 without insulin (Sigma, St Louis, MO, USA). DRG neurons from control rats were cultured in the presence of 5 mM D-glucose and DRG neurons derived from STZ-induced diabetic rats with 25 mM D-glucose. No neurotrophins or insulin was added to any DRG cultures unless otherwise indicated. The HEK293 (ATCC CRL-1573, Virginia, USA) cell line was cultured in DMEM/F12 (1:1) media containing 10 mM glucose and 10% FBS. Sorbinil, a selective inhibitor of aldose reductase was purchased from a commercial supplier (Sigma, St Louis, MO, USA). For more details, see Supplementary Information.

The human neuroblastoma-derived cell line SHSY5Y (ATCC CRL-2266; ATCC, Gaithersburg, MD, USA), a popular albeit imperfect neuronal model, was cultured and expanded for experiments in 42% Ham’s F12 media (Sigma Aldrich), plus 42% EMEM media (Sigma Aldrich), 15% fetal bovine serum, 1% penicillin-streptomycin, and 1% non-essential amino acids, with enzymatic passaging using TrypLE Select (Thermo Fisher Scientific). SHSY5Y neural cells were plated onto 96-well culture plates at 25,000 cells/well and grown for 1–2 days before treatments were applied.