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3 protocols using iron 3 chloride hexahydrate

1

PHBV/PVA Composite Biofilm Preparation

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PHBV with a PHV content of 12 wt% was purchased from Goodfellow (Huntingdon, UK). Polyvinyl alcohol (PVA) (MW ~30,000), Ringer´s solution and Iron (II) chloride tetrahydrate were from Merck (Darmstadt, Germany). DCM, propidium iodide (PI), sodium citrate, triton X-100, acetone and lauric acid were obtained from Sigma-Aldrich (St. Louis, USA). Ammonia solution 25% and Iron (III) chloride hexahydrate were supplied by Roth (Karlsruhe, Germany). TCH was purchased from AppliChem (Darmstadt, Germany). RPMI 1640 medium, fetal calf serum (FCS), Annexin A5-FITC (Ax), Hoechst 33342 (Hoechst) and DiIC1 (5) were acquired from Thermo Fisher Scientific (Waltham, USA).
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2

Synthesis and Characterization of Iron Nanoparticles

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Iron(II) chloride tetrahydrate (FeCl2⋅4H2O), hydroxylammonium chloride, bovine serum albumin (BSA), and Eppendorf ultrafiltration tubes with a molecular weight cutoff (MWCO) of 3 kDa were purchased from Merck (Darmstadt, Germany). Iron(III) chloride hexahydrate (FeCl3⋅6H2O), dialysis tubes (Spectrapor 6, MWCO 8 kDa), ammonium chloride, formic acid, hydrochloric acid 25 %, ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), sodium thiosulfate, silver nitrate, sodium carbonate, and ammonia solution 25 % were supplied by Roth (Karlsruhe, Germany). Propidium iodide (PI), sodium citrate, triton X-100, ninhydrin, MES, HEPES, and poly(acrylic acid-co-maleic acid) solution (Mw = 3000 Da) were purchased from Sigma-Aldrich (St Louis, MO, USA). Ringer’s solution was bought from Baxter Healthcare (Zurich, Switzerland). Annexin V-FITC (AxV), Hoechst 33342 (Hoe), and DiIC1(5) (hexamethylindodicarbocyanine iodide dye [DiI]) were purchased from Thermo Fisher Scientific (Waltham, MA, USA).
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3

Fluorescent Labeling of Annexin A5

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Iron (II) chloride tetrahydrate (FeCl2·4H2O), hydroxylammonium chloride, bovine serum albumin (BSA), and Eppendorf ultrafiltration tubes with a molecular weight cutoff (MWCO) of 3 kDa were purchased from Merck (Darmstadt, Germany). Iron (III) chloride hexahydrate (FeCl3·6H2O), dialysis tubes (Spectrapor 6, MWCO 8 kDa), ammonium chloride, formic acid, hydrochloric acid 25%, and ammonia solution 25% were supplied by Roth (Karlsruhe, Germany). Propidium iodide (PI), sodium citrate, triton X-100, ammonium formate, lauric acid, and acetone were purchased from Sigma-Aldrich (St Louis, MO, USA). Sulfosalycylic acid solution 20% was bought from Applichem (Darmstadt, Germany). Mitoxantrone (MTO) solution (2 mg/mL) was purchased from TEVA Pharma (Ulm, Germany). Ringer’s solution was bought from Baxter Healthcare (Zurich, Switzerland). Falcon ultrafiltration tubes (MWCO 100 kDa) were purchased from Sartorius (Goettingen, Germany). Recombinant chicken Annexin A5 (AxA5; responsif GmbH, Erlangen, Germany) was labeled with fluorescein isothiocyanate (AxA5-FITC) according to the manufacturer’s instructions (Sigma-Aldrich). Hoechst 33342 (Hoe) and DiIC1(5) (hexamethylindodicarbocyanine iodide dye [DiI]) were purchased from Thermo Fisher Scientific (Waltham, MA, USA).
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