Epinephrine

SILAC labelling was performed as previously described [15] (link), [16] (link), briefly: NHF were cultured and passaged in SILAC-DMEM with 4.5 g/L glucose, sodium pyruvate and 3.7 g/L NaHCO₃ (PAN Biotech), supplemented with 10% dialyzed FBS (Gibco), 1% penicillin/streptomycin, 82 mg/L l-proline, 84 mg/L l-arginine HCl (Arg0) and 146 mg/L l-lysine HCl (Lys0) for light labelling (Sigma-Aldrich). For medium-heavy labelled cells, Arg0 and Lys0 were replaced by l-arginine-¹³C₆¹⁴N₄ and l-lysine-²H₄ (Arg6, Lys4). For heavy labelling, l-arginine-¹³C₆¹⁵N₄ and l-lysine-¹³C₆¹⁵N₂ (Arg10, Lys8) were used.
SCC13 cells were cultured in SILAC-Keratinocyte Growth Medium 2 (KGM2) without calcium, l-arginine, and l-lysine (PromoCell), supplemented with SupplementMix containing 0.004 mL/mL bovine pituitary extract, 0.125 ng/mL epidermal growth factor, 5 μg/mL insulin, 0.33 μg/mL hydrocortisone, 0.39 μg/mL epinephrine, 10 μg/mL transferrin and 0.06 μM CaCl₂ (PromoCell), with 1% penicillin/streptomycin. l-Lysine HCl concentrations were the same as mentioned above. For l-arginine HCl, 210 mg/L were used [17] (link).

Human nasal epithelial cells (HNEpC; PromoCell) were cultured in Airway Epithelial Cell Growth Medium (basal medium supplemented with BPE, EGF, insulin, hydrocortisone, epinephrine, triiodo-L-thyronine, transferrin and retinoic acid; PromoCell) at 37 °C in a 5% CO₂ atmosphere. HNEpC cells at passage 5 were used in the experiments. The cells were plated in 48-well plates (BD Falcon) coated with type I collagen (Corning) at the initial density of 1 × 10⁴ cells/cm², 24 h prior to the experiments. The growth medium was then changed to the experimental cell culture medium containing nanoparticles or vehicle control (water). The experimental medium consisted of phenol red-free DMEM, prepared from powder using water or a mixture of water and 214 mg L⁻¹ TA-AgNP suspension in water and supplemented with 1 gL⁻¹ glucose, NaHCO₃ (Gibco), sodium pyruvate (Lonza), 1% bovine serum albumin (BSA), L-glutamine and P/S (50 units/mL of penicillin and 50 µg/mL of streptomycin). Water (used as vehicle control) was a component of the cell culture medium, not an additive to the medium, and as such, the osmolality of the experimental medium was not compromised. A 48 h incubation in the experimental medium had no adverse effects on the viability of the cells compared to the PromoCell culture medium. Cell culture reagents were from Sigma unless otherwise specified.

We cultivated primary human skin fibroblast lines (HF40 and HFF-1) (n = 2 each) that were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultivated in Dulbecco's minimum essential medium supplemented with 10% fetal calf serum and, when confluent, medium was supplemented with or without recombinant human (rh)-IL-17 (R&D System, Minneapolis, MN) of 200 ng ml⁻¹ (same IL-17 source and concentration used in prior experiments with human keratinocytes) [10] (link), [12] (link). After 24-hour incubation, fibroblasts were harvested for further analyses.
We also cultivated NHEKs obtained from PromoCell, in the Keratinocyte Growth Medium 2 supplemented with 0.004 ml/ml BPE, 0.125 ng/ml EGF, 5 ug/ml Insulin, 0.33 ug/ml Hydrocortisone, 0.39 ug/ml Epinephrine, 10 ug/ml Transferrin, and 0.06 mM Ca++ (all items purchased from PromoCell GmbH, Heidelberg, Germany). The experiment was performed in triplicate.
Once 70–80% confluent, the medium was changed with full media containing 0.06 mM Ca++, 1.2 mM Ca++, or 1.2 mM Ca++ plus 2.0% FBS, for 24 and 48 hours before harvesting for other analyses.