Amphotericin b

Human keratinocytes were isolated from neonatal foreskin as previously described [35 (link)]. PHK were cultured in Keratinocyte-SFM (Thermo Fisher Scientific, Waltham, MA, USA) with 1% Pen/Strep, 0.2% Amphotericin B (Thermo Fisher Scientific, Waltham, MA, USA). To differentiate PHK, cells were grown in DMEM (11965092, Thermo Fisher Scientific, Waltham, MA, USA) with 1% Pen/Strep, 0.2% Amphotericin B. For cytokine experiments, the following reagents were added alone or in combination to the culture media from the time of differentiation and replaced with every 48-h media change: human IL-4 (5–50 ng/mL; R&D system, Minneapolis, MN, USA), human IL-13 (5–50 ng/mL; R&D system, Minneapolis, MN, USA), human IL-17A (1–100 ng/mL; R&D system), JAK inhibitor I (10 μM; Calbiochem, San Diego, CA, USA), and PD98056 (10 μM; Calbiochem, San Diego, CA, USA). For the epidermal organotypic model experiment, keratinocytes were grown as previously described [36 (link)]. For all cytokine treatment studies, keratinocytes were starved of growth factors for 24 h before stimulation with the indicated cytokines.

Ethical approval is granted and patients gave consent with the usage of the tumour samples to isolate of human primary schwannoma cells. The methods were described by Rosenbaum et al.^{58 (link)} Briefly, schwannomas were surgically removed under local anaesthesia, and were then preincubated for 1–7 days in incubation medium (DMEM plus 10% FBS, 500 U/ml penicillin/streptomycin, 0.5 μM Forskolin, 2.5 μg/ml Amphotericin B) in 10% CO₂ and then dissected into 1-mm-long pieces in DMEM with 10% FCS containing 500 U/ml penicillin/streptomycin, 160 U/ml collagenase type I (Sigma) and 1.25 U/ml dispase grade I (Roche, West Sussex, UK). Tissue pieces were incubated in proteolytic enzymes for 24 h before they were dissociated by trituration with a narrowed Pasteur pipette. Cell suspension was added to a 50-ml Falcon tube. Cells were collected and resuspended in proliferation medium: DMEM with 10% FCS, 500 U/ml Pen/Strep, 0.5 μM Forskolin (Tocris, Abingdon, UK), 2.5 μg/ml Amphotericin B, 10 nM b1-heregulin (R&D System, Abingdon, UK) and 2.5 μg/ml insulin (Sigma). Cells were seeded into 96-well plates (Greiner Bio-one, Stonehouse, UK), coated with 1 mg/ml poly-l-lysine (Sigma) and 4 μg/ml natural mouse laminin (Life Technologies, Paisley, UK), at a density of 3000 cells/well. Proliferation medium was changed every 3–4 days and cells were passaged when confluent.

The immortalized rat RT4-D6P2T Schwann cell line (ATCC, Cat #CRL-2768, Manassas, VA) and S16 Schwann cell line (ATCC, Cat #CRL-2941, Manassas, VA) were aseptically cultured and subcultured (at 80% confluency) in Dulbecco’s Modified Eagle Medium (DMEM) (ATCC, Cat #30–2002, Manassas, VA) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher, Cat #16000044, Waltham, MA) and 1% penicillin/streptomycin (Pen-strep) (GIBCO, Cat #15140–015, Gaithersburg, MD)/amphotericin B (R&D Systems, Cat #B23192, Minneapolis, MN) at 37°C and 5% CO₂ in poly-L-lysine-coated dishes. For all cells treated with LPS, lipopolysaccharide (LPS) from Escherichia coli O111:B4 (Sigma, Cat #L2630, St. Louis, MO) was reconstituted in 1 mL of autoclaved deionized water for a concentration of 1 mg/mL and further diluted depending on the required dose for each experiment.