A92902

In order to determine the reduction of oxygen saturation of medium, we employed 96-well microplates Oxoplates^® with integrated chemical optical oxygen sensors (Pressens, Regensburg, Germany). Oxygen saturated medium with and without 100µM ascorbate (Sigma-Aldrich, A92902, Taufkirchen, Germany) was incubated with the indicated concentrations of Co²⁺ for 1 hour. The percentage of oxygen saturation was calculated according manufacturer instructions after measuring the fluorescence from two filter pairs for the indicator (excitation: 535 nm, emission: 680 nm) and reference dye (excitation: 540 nm, emission: 590 nm).

GFPtpz-collagen BMDMs were seeded on matrigel-coated coverslips at a cell density of 3 × 10⁴ cells/cm² together with murine L929 cells (1.5 × 10⁴ cells/cm², obtained from the American Type Culture Collection). In order to stimulate collagen deposition, growing medium (DMEM with 10% FBS, 2 mM L-glutamine and 2% penicillin/streptomycin) was supplemented with a combination of supernatants (SNTs) from stimulated L929 fibroblasts and Mouse primary Cardiac microvascular Endothelial Cells, MCEC, (C57-6024, obtained from Cell Biologics) in a ratio of 2:1:1 (growing medium:L929 SNT:MCEC SNT) during 6 to 9 days. To facilitate collagen deposition, 50 µg/ml of fresh ascorbic acid (A92902, Sigma-Aldrich) was added daily and medium supplemented with SNTs was changed every 2 days. For supernatants preparation, L929 and MCEC monoculture were grown to confluence in DMEM supplemented with 10% FBS, 2 mM L-glutamine and 2% penicillin/streptomycin. L929 cells medium was supplemented with 10 ng/ml TNFα (410-MT/CF, R&D Systems), 10 ng/ml IL-1β (401-ML/CF, R&D Systems), 10 ng/ml IL-4 (404-ML/CF, R&D Systems) and 10 ng/ml TGFβ (7666-MB/CF, R&D Systems). MCEC medium was supplemented with 10 ng/ml TNFα (410-MT/CF, R&D Systems). After 48 h of incubation, the supernatants from both monocultures were harvested, centrifuged to remove cell debris and stored at −20 °C until use.

HFL-1 (CCL-153; ATCC, Manassas, VA, USA), IMR-90 (CCL-186; ATCC), and WI-38 (CCL-75; ATCC) human lung fibroblasts and A549 human lung adenocarcinoma cells (CCL-185; ATCC) were used in this study. HFL-1 was cultured in F12–K medium (21127022; Thermo Fisher Scientific, Waltham, MA, USA). IMR90 and WI-38 cells were maintained in Eagle's minimum essential medium (10-009-CV; Corning Cellgro, Manassas, VA, USA). A549 cells were cultured in the Roswell Park Memorial Institute 1640 medium (10-040-CVRC; Corning Cellgro). All media were supplemented with 10% fetal bovine serum (16000044; Thermo Fisher Scientific) and 1% antibiotic-antimycotic (15240062; Thermo Fisher Scientific). Cells were grown in 5% CO₂ at 37 °C. VitC (l-ascorbic acid; A92902; Sigma-Aldrich, St. Louis, MO, USA) and NAC (A9165; Sigma-Aldrich) were dissolved in distilled water, vitamin D (VitD) (Cholecalciferol; C9756; Sigma-Aldrich) was dissolved in ethanol, and vitamin E (VitE) (DL-α-Tocopherol acetate; T3376; Sigma-Aldrich) was dissolved in chloroform, according to the manufacturer's instructions.