Phosphate buffered saline solution pbs

Type A gelatin from porcine skin with a high molecular weight and Bloom number of 300, polyethylene glycol diacrylate (PEGDA, Mn = 700 g mol⁻¹), phosphate buffered saline solution (PBS; pH = 7.4) were obtained from Sigma-Aldrich (Saint-Louis, MO, United States). The chemicals were used as received without additional purification.

The experimental procedures were approved by the South Australian Health and Medical Research Institute Animal Ethics Committee and followed the guidelines of the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes developed by the National Health and Medical Research Council. The investigators understood the ethical principles outlined in Grundy et al.^{26 (link)}. Adult pregnant ewes (4 yrs old; n = 4) were housed in an individual pen in view of other sheep in an indoor housing facility that was maintained at a constant ambient temperature of between 20–22 °C and a 12 hour light/dark cycle. Sheep were humanely killed via overdose of sodium pentobarbitone (8 g; Vibrac Australia, Peakhurst, Australia). Samples of cardiac muscle tissue were taken from the left ventricle and skeletal muscle was taken from the quadriceps. Collected tissue samples were placed into sterile phosphate-buffered saline solution (PBS; Sigma-Aldrich, St. Louis, USA) on ice, protected from light, transported to the imaging facility within 90 minutes and imaged within 7 hours. All experiments were performed on samples from a minimum of 4 animals on 3 separate days.

ε-Caprolactone (CL, Acros, 99% purity), 2-ethylhexanoic acid tin(II) salt (Sn(Oct)₂; Sigma-Aldrich, St. Louis, MO, USA, purity ≥95%), 4,4′-Azobis(4-cyanovaleric acid) (ACVA; Sigma-Aldrich, purity ≥ 98%), 4-Cyano-4-(phenylcarbonothioylthio)pentanoic acid (CPA; Sigma-Aldrich, purity ≥ 97%), poly(ethylene glycol) methyl ether methacrylate (PEGMA; Sigma-Aldrich, M_n 950 Da), Tween80 (Sigma-Aldrich), Diethyl ether (DEE, Sigma-Aldrich, ≥99%), Deuterated chloroform (CDCl₃, Sigma-Aldrich, 99.8%), Ethanol (EtOH, Sigma-Aldrich, 99.8%), acetonitrile (ACN, Sigma-Aldrich, 99.9%), 6-hydroxycaproic acid (HCA, ABCR, Karlsruhe, Germany, 95%) were used as received without further treatments. The cell medium is composed by high glucose DMEM/F12 (Biowest, Nuaillé, France) supplemented with 10% fetal bovine serum (Lonza, Basel, Switzerland). Phosphate-buffered saline solution (PBS) was purchased from Sigma-Aldrich. For gel permeation chromatography (SEC) analysis Tetrahydrofuran (THF, Sigma-Aldrich, purity ≥ 99.7%) was used as eluent.

Human iris tissue was processed as described [29 (link),30 (link)]. Briefly, iris tissue was treated with 0.2% collagenase (Sigma-Aldrich, St. Louis, MO, USA) and washed twice with phosphate-buffered saline solution (PBS; Sigma). The isolated cells were cultured in iris culture medium (iris medium): Advanced Dulbecco’s Modified Eagle Medium/Ham’s F12 (Advanced DMEM/F12; ThermoFisher Scientific, Waltham, MA, USA) supplemented with 5% mixed serum (heat-inactivated human serum [Sigma], KnockOut™ Serum Replacement [ThermoFisher Scientific], and Artificial Serum, Xeno-free [Cell Science & Technology Institute, Miyagi, Japan] in a 5:3:2 ratio), 10 ng/mL basic fibroblastic growth factor (b-FGF; Sigma), 10 ng/mL epidermal growth factor (EGF; Sigma), ×50 GlutaMAX (Gibco Invitrogen, Carlsbad, CA, USA), CultureSure^® Y-27632 solution (only for the beginning of the culture; Fujifilm Wako Pure Chemical Corp., Osaka, Japan), and 1% penicillin/streptomycin (Sigma) in a 35 mm culture dish coated with collagen type-1 (Toyobo, Osaka, Japan) at 37 °C in a 5% CO₂ humidified incubator.

Recombinant human IL-1β was purchased from Sigma–Aldrich s.r.l. (Milan, Italy). Human phosphor-VEGFR-2 polyclonal antibody was purchased from R&D Systems (Minneapolis, MN, USA). Flexi Tube GeneSolution, consisting of four pre-selected siRNAs for ESM-1 (endocan siRNA) to induce endocan knockdown, the negative control siRNA, and the transfection reagent (HiPerFect Transfection Reagent) were supplied by Qiagen (Hilden, Germany). Basal chondrocyte medium, fetal bovine serum (FBS), penicillin/streptomycin mixture, and glutamine were obtained from SCIENCELL™ (Carlsbad, CA, USA). Phosphate buffered saline solution (PBS) and other general laboratory chemicals were obtained from Sigma-Aldrich S.r.l. (Milan, Italy).

Growth factors: bFGF (recombinant human fibroblast growth factor-basic; PeproTech) and EGF (recombinant human epidermal growth factor; PeproTech), DMEM/F-12 medium (1 : 1) and Neural Basal medium (Gibco/Life Technologies), glucose at 30% (w/v) in distilled water (ThermoFisher Scientific) and stored at 4°C (d-(+)-glucose, Sigma-Aldrich, Australia), GlutaMAX™-1 (Gibco/Life Technologies), phosphate-buffered saline solution (PBS) (Sigma-Aldrich, Australia) made according to the manufacturer's instructions, poly-d-lysine hydrobromide (Sigma-Aldrich, Australia) reconstituted in double distilled water at 1 mg ml⁻¹ and stored at 20°C, laminin supplied reconstituted at 1 mg ml⁻¹ Tris–HCl (50 mM, pH 7.4), NaCl (0.15 M) and stored at 20°C (Invitrogen/Life Technologies), ITS solution (insulin/transferrin/selenium-A solution, Gibco/Life Technologies), N-2 supplement (Gibco/Life Technologies), B27 retinol-free supplement (Gibco/Life Technologies) and Pen/Strep solution (penicillin/streptomycin solution with 10 000 U ml⁻¹ penicillin and 10 000 mg ml⁻¹ streptomycin, Gibco/Life Technologies).

The subjects inhaled 10 mL of sterile hypertonic saline solution (3%, 4%, or 5% NaCl, Ivex Pharmaceuticals, USA) at room temperature from an ultrasonic nebulizer (DeVilbiss Health Care, USA). The duration of each inhalation was 7 min, and it was stopped after expectoration an adequate amount of sputum. In order to detect a possible decrease in FEV₁, spirometry was performed after each inhalation. Sputum was poured into a Petri dish and separated from saliva. A 4-fold volume of freshly prepared 0.1% dithiothreitol (DTT; Sigma-Aldrich) was added. The mixture was vortexed and placed on a bench rocker for 15 min at room temperature. Next, an equal volume of phosphate-buffered saline solution (PBS; Sigma-Aldrich) was added to DTT. The cell pellet was separated using a 40-μm cell stainer (Becton Dickinson, USA). The mixture was centrifuged for 10 min at 4°C; the supernatant was aspirated and stored at −70°C for later analysis. The total cell counts, percentage of epithelial cells, and cell viability were investigated using a Neubauer hemocytometer (Heinz-Herenz, Germany) under a microscope (B5 Professional, Motic, China) by employing the Trypan blue exclusion method. The cytospin samples of induced sputum were prepared using a cytofuge instrument (Shandon Southern Instruments, USA).