Human total mmp 1 duoset

NHDF cells were seeded in 96-well plates at a density of 10⁴ cells/well in DMEM-DCC-FBS medium. Twenty-four hours later, cells were pre-incubated with phytonutrients or estradiol for 24 h. Vehicle-treated control cells were incubated with the relevant amounts of solvents used in a particular experiment, which had no effect on the measured parameters. The medium was then replaced with one containing the treatment compounds, with or without H₂O₂ and incubated for an additional 24 h. Thereafter, medium was removed and frozen for the analysis of secreted proteins, and the cell number was determined by the XTT Cell Proliferation Kit (Biological Industries, Beth Haemek, Israel), according to the manufacturer’s instructions. MMP-1 and pro-collagen 1a1 protein levels in cell culture supernatants were quantified by ELISA using the Human Total MMP-1 DuoSet and Human Pro-Collagen 1a1 DuoSet, ELISA kits (R&D Systems, Inc., Minneapolis, MN, USA), according to the manufacturer’s instructions. Optical density was measured using a VERSAmax tunable microplate reader (Molecular Devices, Menlo Park, CA, USA). Results of the cell number, MMP-1, and pro-collagen were calculated as a percent of the values obtained in control cells, treated with a vehicle without H₂O₂.

MMP-1 protein levels in cell culture supernatants were quantified by ELISA using the Human Total MMP-1 DuoSet (cat# DY901B, R&D Systems) according to manufacturer’s instructions. The detection limit was 62.5 pg/mL. Signal saturation was observed at concentrations > 4000 pg/mL. The optical density was measured using a Varioskan Flash multimode reader (Thermo Scientific, USA) at a wavelength of 450 nm. Averages of the optical densities obtained from the duplicate readings for each standard, control, and sample were calculated. MMP-1 concentrations were then calculated by generating a four-parameter logistic (4-PL) curve-fit.

The cultured HDFs were starved in serum-free DMEM for 24 h and treated with 100, 200, or 400 μg/mL of OHE in serum-free media for 1 h, followed by sUV (23 kJ/cm²) radiation. After 48 h, the supernatants were collected and analyzed with Human Total MMP-1 DuoSet and Human Pro-collagen I alpha 1 DuoSet ELISA kits (R&D Systems, Inc., Minneapolis, MN, USA), according to the manufacturer’s protocols.