Mithras multimode reader

All POPs were tested in the presence of 0.04 nM 17β-estradiol used as a positive control (PC), calculated as the EC50 concentration of 17β-estradiol standard curve (Figure . 2). Cells were exposed to the concentration range of POPs given in Table 1. DMSO was used as a J o u r n a l P r e -p r o o f solvent control (SC) (0.2% v:v in assay media). Following 48 h exposure, the supernatant was discarded, and the cells washed twice with PBS, lysed with 1 × cell lysis reagent facilitated by agitation (Promega, Southampton, UK). Then, 100 µL of Luciferase (Promega, Southampton, UK) was injected into each well, and Luciferase activity was measured using the Mithras Multimode Reader (Berthold, Germany). The response of the cell line to the POPs was measured and compared with the PC (0.04 nM 17β-estradiol).

RGAs were carried out as previously described by Frizzell et al. (2011) . Briefly, cells were seeded at a concentration of 4 × 10 5 cells/ml, 100 μl/well, into white walled 96 well plates with clear flat bottoms (Greiner Bio-One, Germany). The cells were incubated for 24 h and then exposed to BEA and FB1 (0.001, 0.01, 0.1, 1, 10 μM) for the agonist test. The positive control used with each cell line was as follows: 1.35 ng/ml progesterone (TM-Luc cells) and 181 ng/ml hydrocortisone (TGRM-Luc cells). A solvent control 0.5% (v/v) methanol in media was also added to each plate. Antagonist tests were carried out by incubating BEA and FB1 (0.001, 0.01, 0.1, 1, 10 μM) with the relevant positive control for each cell line. The cells were incubated for 48 h, after which, the media was discarded and the cells washed once with phosphate buffered saline (PBS). The cells were lysed with 30 μl cell culture lysis buffer (Promega, Southampton, UK) and then 100 μl luciferase (Promega, Southampton, UK) injected into each well and the response measured using the Mithras Multimode Reader (Berthold, Other, Germany). The response of the cells to the various compounds was measured and compared with the solvent control.