Elisa reader

Circulating leptin levels were measured using a commercial ELISA kit following manufacturer's instructions (Enzo Life Sciences, Farmingdale, NY). Plates were read at 450 nm in ELISA reader (Dynex Technologies, USA). Lyophilized leptin ranging from 31.3 to 2000 pg/ml was used to construct the standard curve.

The treatment-induced inhibition of cell proliferation was assessed using crystal violet staining, as described earlier [22 (link)]. In brief, 1500 cells/well seeded in 96-well plates were allowed to adhere to the bottom of the wells for 72 h. Thereafter, the cells were incubated with rising concentrations (0.1–20 μM) of each test compound for up to 48 h. After that, cells were rinsed with PBS, fixed with 1% glutaraldehyde, and 0.1% crystal violet (N-hexamethylpararosaniline, Sigma Aldrich) was added to stain the cells. Unbound dye was removed by rinsing with water. The cell-bound crystal violet was dissolved using 0.2% Triton X-100 (Sigma-Aldrich, Munich, Germany). The extinction of crystal violet, which increases linearly with the increase of the cell number, was measured with an ELISA-Reader (Dynex Technologies, Denkendorf, Germany) at 570 nm [23 (link)].

PGE₂ level in BALF supernatants was measured using commercially PGE₂ high-sensitivity ELISA kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's protocol. Optical density was determined on an ELISA reader (Dynex Technologies, UK) at 450 nm and with a wavelength correction at 570 nm. Each well was assayed in duplicate and averaged. With the generation of a four-parameter logarithmic curve, the concentration in ng/ml PGE₂ in the BALF could be determined.

To observe calcium deposition, cells were fixed with 4% paraformaldehyde at room temperature for 20 min and stained with 2% alizarin red S solution (pH 4.2, adjusted with 1.0% NH₄OH) for 10 min at room temperature. After staining, calcium deposits were photographed. After imaging, stained cells were destained with 10% acetic acid for 20 min, and the calcium concentration was measured based on the absorbance at 420 nm using an ELISA reader (Dynex, Lincoln, UK).

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to assess proliferation of the PASMCs. In brief, after serum starvation for 48 h, proliferation of the PASMCs was induced by PDGF-BB (20 ng/mL) in DMEM supplemented with 1% FBS in 96-well plates at a density of 2 × 10⁴ cells per well, with or without 1–10 nM liraglutide (Novo Nordisk, Novo Alle, Bagsvaerd, Denmark) pre-treatment (1 h). The GLP-1 receptor antagonist 500 nM exendin (9–39) (Sigma–Aldrich, St. Louis, MO, USA) was added to the cells for 30 min prior to treatment with liraglutide. After induction for 24 h, 48 h, and 72 h, MTT (0.5 mg/mL) was added to the medium for 4 h. The culture medium was then removed, and the cells were dissolved in isopropanol and shaken for 10 min. The amount of MTT formazan was quantified at 540 and 630 nm using an enzyme-linked immunosorbent assay (ELISA) reader (DYNEX Technologies, Denkendorf, Germany).

Cellular growth assays were conducted using crystal violet dye elution procedure as described previously^{43 (link)}. In brief, HCEC were seeded at a density of 1–1.2 × 10⁴ cells/cm² in 96 well plates, and after 24 h of growth, 1/10 dilution of assayed products (CBED and AMED) was added. After 24, 48 and 72 h of incubation, cell cultures were washed and incubated with a 0.25% solution of crystal violet in ethanol for 20 min. The dye was eluted for 30 min in 100 µL of 33% acetic acid. Absorbance was measured at 590 nm using an ELISA reader (DYNEX Technologies, VA, USA). Three independent experiments (using 3 different donors) were carried out in triplicate (3 wells).