Clear flat bottoms

MMV-Luc cells were seeded at a density of 40,000 cells per well, into white-walled 96 well plates with clear flat bottoms (Greiner Bio-One, Frickenhausen, Germany). Plates were incubated at 37 °C and 5% CO2 overnight prior to exposure to the tested compounds.

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.

MMV-Luc cells were cultured in DMEM (without phenol red) containing 10% charcoalstripped foetal bovine serum without antibiotics and maintained in a 75 cm 2 cell culture flask (Nunc, Roskilde, Denmark) at 37 °C in a humidified atmosphere (95%) with 5% CO2 for at least 48 h before use to ensure that the cells are completely free from hormonal induction from the media. For experiments, attached cells were liberated from flasks using TrypLE™ Express trypsin (Invitrogen TM Life Technologies, Paisley, UK), counted for viability checks prior to seeding plates by trypan blue staining and using a Countess ® automated cell counter (Invitrogen TM Life Technologies, Paisley, UK). Cells (100 µL) were seeded at a density of 4 x 10 5 cells/mL into each well of white walled 96-well plates with clear flat bottoms (Greiner Bio-One, Frickenhausen, Germany) and incubated for 24 hours to allow cells to attach before chemical treatment.