2.4.2. Collagen Synthesis

NHDF cells were homogeneously seeded in 24-well plates at a density of 6 × 10⁴ cells per well and incubated at 37 °C, with a 5% CO₂ humidified atmosphere. After 24 h, the medium was replaced by one with a low FBS concentration (0.5%) as recommended in the kit. After 24 h, the two concentrations of the product (1.25 and 2.5 mg/mL), demonstrated to be non-cytotoxic and having the best solubility from the preliminary MTT so were chosen to be used in this assay (data not shown). Occurring at the end of the treatment, the supernatant of each sample was collected into a sterile tube and incubated overnight with the Isolation and Concentration Reagent (polyethylene glycol TRIS-HCl buffer, pH 7.6). The day after, the measurement of collagen synthesis was performed using a commercial kit (Sircol, Soluble Collagen assay kit, Biocolor Life Science Assays, Carrickfergus, UK), according to the manufacturer’s instructions, and the absorbance of samples was measured at 555 nm. The concentration of collagen then was calculated using a standard curve.