Hank s balanced salt solution hbss

The corrosion behaviour of the SLM-processed Mg-based alloy was analysed using two methods. The first applied was the immersion test for measuring the evolved hydrogen evolution after 4 days of immersion; this test has been carried out in 60 mL Hanks’ Balanced Salt Solution (HBSS, Sigma Aldrich, Merck KGaA, Darmstadt, Germany) at room temperature. The second applied method was the electrochemical corrosion test.
The Autolab Potentiostat/Galvanostat PGSTAT302N (Metrohm, Herisau, Switzerland) and a commonly used cell system with three-electrode were used for the electrochemical corrosion tests. For this system, the testing material was the working electrode, a silver/silver chloride (Ag/AgCl 3M) electrode was employed for the reference electrode, and a platinum one for the counter. Hanks’ Balanced Salt Solution (HBSS, Sigma Aldrich, Merck KGaA, Darmstadt, Germany), pH of 7.0–7.4, was used as electrolyte at room temperature, and Potentio-Dynamic Polarisation tests (PDP) were performed retrieving current-voltage characteristics in a linear sweep mode. The testing parameters were as follows:

Open Circuit Potential (OCP) stabilisation time of 15 min to ensure stable OCP before PDP tests;

Polarization range of ±250 mV;

Scanning rate of 1 mV/s;

E_corr and j_corr for the Tafel method of extrapolation;

Tests performed in triplicate.

Corneal preparations were carried out as previously described (Begum et al., 2020 (link)). Briefly, porcine eyes were procured un-scalded and within two hours of death from Dissect Supplies (Birmingham, UK). Corneas were dissected from the globe and a 5 mm biopsy punch (Stiefel^®, Brentford, UK and WellTech Rapid-Core, Taiwan, China) used to obtain approximately four corneal discs per cornea, which were each fitted separately into a CellCrown 96 well insert (Sigma-Aldrich, Gillingham, UK) with the epithelium facing up. The insert was placed in a black, clear F-bottom 96 well plate (Grenier Bio-one, Stonehouse, UK) that had been prefilled with 100 µL of Hanks’ Balanced Salt Solution (HBSS; Sigma-Aldrich, Gillingham, UK). Thirty microliters of HBSS was added over the corneal epithelium of control wells while 30 µL of 1 M sodium hydroxide (NaOH; Thermo Fisher, Waltham, MA) was added over the corneal epithelium to induce alkali injury or 30 µL of 1 M sulfuric acid (H₂SO₄; SLS, West Bridgford, UK) was added over the corneal epithelium to induce acid injury. Each solution was applied for 2 min before washing three times with HBSS. Neutral pH was confirmed using a pH strip before subsequent test solution application.

The ARPE-19 cells were starved in a serum-deprived DMEM/F12 media containing 1% FCS and 1% penicillin-streptomycin for 24 hours. For the CellTiter 96 AQueous One Solution Cell Proliferation (MTS) Assay, the cells were exposed to 0.025% dimethyl sulfoxide (DMSO; Sigma Aldrich, USA) as the drug vehicle control or 3µM LSF, 5µM LSF, 10µM LSF, 20µM LSF or 30µM LSF for 24 hours. For the GC-MS/LC-MS analysis, the cells were exposed to 0.025% DMSO, 5µM LSF or 20µM LSF for 24 hours. The negative control for all analyses was untreated cells that were incubated in serum-deprived DMEM/F12 media. The term “untreated cells” refers to cells not treated with LSF regardless of the oxidative stress stimulus being present or not. After 24 hours incubation, the treatments were discarded from all the wells and the cells were incubated with 200µM hydrogen peroxide (H
₂O
₂; Sigma Aldrich, USA) as an oxidative stress stimulus for two hours. Untreated cells or LSF-treated cells incubated in Hanks Balanced Salt Solution (HBSS; Sigma Aldrich, USA) for two hours were used as the negative control for oxidative stress. Subsequently, the H
₂O
₂ or HBSS was removed and the cells were allowed to recover for 24 hours in serum-deprived DMEM/F12 media before either the MTS assay or the GC-MS/LC-MS analysis were carried out
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