Fetal bovine calf serum

For differentiation of 3T3‐L1 cultures to adipocytes, cells were seeded into 96‐well plates (Dutscher Scientific) at a density of 20 000 cells per well in 100 μl maintenance medium. Following a 2 day stabilization period, they were switched to differentiation medium consisting of DMEM supplemented with 2 mm glutamine, 10 μg ml⁻¹ penicillin/streptomycin, 10% fetal bovine calf serum (GE Healthcare Life Sciences), 500 μm 3­isobutyl­1­methylxanthine (Sigma‐Aldrich Ltd) and 100 nm insulin from bovine pancreas (Sigma‐Aldrich Ltd). After a further 2 day incubation, the medium was refreshed but lacked insulin and contained various concentrations of the test neonicotinoids. Treatment with dexamethasone acted as a positive control. Media were replenished every 2 days for a further 6 days. Lipid accumulation on day 8 following induction of differentiation was visualized using fluorescent Nile Red staining in accordance with the manufacturer's instructions (AdipoRed™ Assay Reagent; Lonza, Walkersville, MD, USA) and quantified using a microplate reader (GloMax® Multi Microplate Multimode Reader; Promega, Madison, WI, USA).

For differentiation of murine 3T3-L1 cultures to adipocytes, cells were seeded into 96-well plates (Dutscher Scientific, Brentwood, UK) at a density of 20 000 cells per well in 100 µl maintenance medium. Following a 2-day stabilization period, they were switched to differentiation medium consisting of DMEM, 2 mM glutamine, 10 µg/ml penicillin/streptomycin, 10% fetal bovine calf serum (GE Healthcare Life Sciences, Buckinghamshire, UK), 500 μM 3-isobutyl-1-methylxanthine (Sigma-Aldrich Ltd) and 100 nM insulin from bovine pancreas (Sigma-Aldrich Ltd). After a further 2 days of culture, the medium was refreshed to start the incubation with various concentrations of the test substances. Dexamethasone was used as a positive control. Media were replenished every 2 days for a further 6 days. Lipid accumulation was visualized on day 8 using fluorescent Nile Red staining in accordance with the manufacturer’s instructions (AdipoRed Assay Reagent, Lonza Walkersville Inc, USA) and quantified using a microplate reader (GloMax Multi Microplate Multimode Reader, Promega, Madison). The fluorescence was measured with a filter giving an excitation at 490 nm and emission at 510–570 nm. The adipogenic effect was expressed as a fold change in emission signal intensity between untreated differentiated and treated differentiated 3T3-L1 cells.