Acetic acid

Trolox, 2,4,6,-tri (2-pyridyl)-s-triazine (TPTZ), gallic acid, procyanidin B2 and myrtenol were purchased from Sigma-Aldrich (St. Louis, MO, USA). Hydrochloric acid, acetic acid, methanol, sodium carbonate, iron chloride, ammonium acetate and Folin-Ciocalteu reagent were purchased from Kemika (Zagreb, Croatia). 2,2′-azinobis (3-ethylbenzothiozolinesulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were purchased from Fluka (Buchs, Switzerland). Neocuproine and copper chloride were obtained from Gram-mol (Zagreb, Croatia). Sucrose was purchased from Gram-mol (Zagreb, Croatia), while trehalose was obtained from Hayashibara doo (Okayama, Japan). Citrus fibers were obtained from Biesterfeld AG (Zagreb, Croatia).

Acetic anhydride, acetic acid, sulphuric acid (96%), iron(III) chloride, potassium iodide and mercury(II) chloride were purchased from Kemika (Zagreb, Croatia). Methanol, dichloromethane, ethyl acetate, cyclohexane and potassium peroxodisulphate were obtained from VWR Chemicals (Radnor, PA, USA) while chloroform was obtained from Honeywell (Charlotte, NC, USA). ABTS and linoleic acid were purchased from Alfa Aesar (Kandel, Germany). Trolox^® was obtained from Acros Organics (Geel, Belgium). DPPH, β-carotene and lipoxidase type I-B from soybean were purchased from Sigma-Aldrich (Darmstadt, Germany). Finally, Dulbecco’s modified Eagle medium (DMEM), foetal bovine serum (FBS), L-glutamine, penicillin and streptomycin were obtained from Capricorn Scientific (Ebsdorfergrund, Germany).

To test cell growth characteristics on an extracellular matrix (ECM), Collagen I was used as an ECM representative protein. Collagen I (Sigma Aldrich, St Louis, MO, USA) was dissolved in acetic acid (50 mM, Kemika, Zagreb, Croatia), diluted in redistilled sterile water in a final concentration of 2 mg/mL and used in the native state or modified by 1 or 10 µM HNE (Enzo Life Sciences, Lausen, Switzerland). Depending on the type of analysis, different formats of cell culture dishes were used with the same coating conditions: Native or modified collagen to its final concentration of 5 μg/cm². Thus, coated cell culture dishes were left to dry in a laminar flow cabinet overnight at room temperature (RT) and subsequently sterilized under UV light for 20 min. Dot-blot analysis with HNE-histidine monoclonal antibody was applied to confirm the binding of HNE to Collagen I had occurred (Supplementary Figure S1). After confirmation that HNE did bind to histidine residues of collagen, we proceeded with evaluating the influence of collagen on measured parameters. Cells were also seeded on uncoated surfaces, further referred to as polystyrene (PS).