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3 protocols using sodium desoxycholate

1

Quantifying Cellular Mercury Content

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PBCECs were cultivated in rat tail collagen-coated 24 well culture plates and cultured according to the cultivation in Transwell® filters. After 72 h incubation of the respective Hg species, PBCECs were washed twice with PBS (100 mM NaCl, 4.5 mM KCl, 7 mM Na2HPO4, 3 mM KH2PO4 (all Sigma Aldrich); pH 7.4) and incubated with 120 μL lysis buffer (RIPA-buffer; 0.01 M Tris, pH 7.6, 0.15 M NaCl, 0.001 M EDTA, 1% sodium desoxycholate, 0.1% (all Sigma Aldrich)) for 15 min on ice. After scrapping off and sonication, the suspension of lysed cells was centrifuged at 10 000 x g for 20 min at 4 °C. Total cellular Hg content was quantified by inductively coupled mass spectrometry (ICP-MS; Agilent 8800 ICP-QQQ, Agilent Technologies Deutschland GmbH, Boeblingen, Germany) in an aliquot of the supernatant. The Bradford assay was used to determine the cellular protein level.
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2

Scaffold-based Human Dermal Fibroblast Culture

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Scaffolds were cut, clamped into 11 mm diameter cell crowns and afterwards sterilized by autoclavation. Hdf were seeded on top in a concentration of 30,000 cells per square centimeter. Cell culture medium consisted of dulbecco’s modified eagle medium (DMEM, Gibco® Life Technologies, Carlsbad, CA, USA), containing 10% Fetal Calf Serum (FCS, LOT: BS 210601.5, Bio and SELL GmbH, Feucht, Germany), 1% penicillin/streptomycin (pen/strep, PAA, Coelbe, Germany) and either 100 or 500 µmol L-Ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Asc) (Sigma-Aldrich, Schnelldorf, Germany) for proliferation and ECM synthesis stimulation. Medium was renewed three times a week. Cultivation lasted four weeks under standard cell culture conditions (37 °C, with an amount of 5% CO2 and a relative humidity of 95%). The complete decellularization process was conducted under sterile conditions. Therefore, specimens were rinsed thrice with phosphate buffered saline (PBS-) (Sigma-Aldrich) and incubated in decellularization solution containing 22.5 g/L sodium desoxycholate (Sigma-Aldrich, Schnelldorf, Germany) in deionized (DI) water. The procedure was repeated on the following day. Specimens were stored in pen/strep containing PBS- until further usage.
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3

Western Blot Protein Analysis Protocol

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Protein extracts were prepared using a RIPA buffer (50 mM Tris/HCl (pH 8.0), 0.1% SDS, 0.5% sodium desoxycholate (all from Sigma-Aldrich), 150 mM NaCl (Carl Roth, Karlsruhe, Germany), and 1% Triton X-100 (Roche, Penzberg, Germany)) with 5% protease inhibitor (Sigma-Aldrich). Protein concentrations were determined using the Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA). Samples were diluted to equal concentrations with 1× Roti®-Load 1 (Carl Roth). SDS-PAGE was performed following standard protocols. The Pierce™ Power Blotter (Thermo Fisher Scientific) was used for semi-dry protein transfer to PVDF membranes (Pall, Port Washington, NY, USA). Membranes were blocked with 5% non-fat dried milk (AppliChem, Darmstadt, Germany)/TBS-T (40 mM Tris/HCl (pH 7.6), 273 mM NaCl, 0.1% Tween® 20 (Sigma-Aldrich)) for 1 h at room temperature. Incubation with primary antibodies was performed in blocking solution overnight at 4 °C, and incubation with secondary antibodies was performed in TBS-T for 1 h at room temperature. Immunoblots were developed with SuperSignal West Pico or Femto Chemiluminescent Substrates (Thermo Fisher Scientific), and signals were detected on a ChemiDoc Touch Imaging System (Bio-Rad). Bands were quantified using Image Lab v6 (Bio-Rad). The antibodies and the concentrations used are listed in Supplementary Table S2.
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