Verioskan flash multimode reader

This assay was performed as previously described^{56 (link)}. KGN cells were seeded in 24-well plates and left overnight. On the following day, the medium was replaced with serum-free medium and the cells were pretreated with the test chemicals for 24 h. Testosterone (10 nM) was then added to each well, and cells were incubated for a further 24 h. The culture medium and cell lysate were collected and stored at −20 °C. The levels of 17β-estradiol in the culture medium were quantified by use of a magnetic particle-based 17β-estradiol enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions (Bio-Ekon Biotechnology, Beijing, China). The intensities were measured at 550 nm with a Verioskan Flash Multimode Reader (Thermo Scientific, Waltham, MA) and normalized to the total cellular protein content. Protein determination was conducted with a bicinchoninic acid (BCA) protein assay kit (Bestbio, Shanghai, China).

UMR106 cells seeded in 6-well plates overnight were treated with 14 and sildenafil for the indicated time. The pre-cooled PBS buffer (120-150 μL) was added in 1×10⁶ cells to keep the cells suspended. The cells were lysed with the repeated freeze-thaw process. After centrifugation for 10 min at 1500 × g at 2-8°C, the supernatants were collected to carry out the assay. The cGMP concentration was determined with a commercial cGMP enzyme immunoassay kit (Elabscience, Wuhan, China). Thereafter, the results were measured with Thermo Scientific Verioskan Flash Multimode Reader at a wavelength of 450 nm ± 2 nm.

The cell-based estrogen biosynthesis assay was performed as previously described [19] (link). Briefly, KGN cells were seeded in 24-well plates overnight. The following day, the cells were replaced with serum-free DMEM/F-12 medium and pretreated with the test chemicals for 24 h. Testosterone (10 nM) was then added to each well, and the cells were incubated for a further 24 h. The culture media and cell lysates were collected and stored at –20°C. The 17β-estradiol in the culture medium was quantified using a 17β-estradiol magnetic particle-based enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions. The optical density (OD) value was measured at 550 nm with the Verioskan Flash Multimode Reader (Thermo Scientific, Waltham, MA). The results, normalized to the total cellular protein content, were expressed as percentages of the control.