Pgl4.53 pgk

Untreated placental 3A cells were trypsin digested and transferred into 6-well plates at low confluency (≤40%). After allowing the cells to adhere to the plate (14–18 hours), cells were transfected using a mixture of 600ng haplotype promoter plasmid DNA, 66ng firefly luciferase control plasmid pGL4.53 PGK (Promega, Madison, WI) and 2μL Lipofectamine 3000 transfection reagent (Invitrogen, Carlsbad, CA). After transfection, cells were allowed to recover for 36–48h and were then exposed to BPA, BPS or a mixture of both bisphenols dissolved in 3 × 10⁻⁵ % ethanol. For the initial acute exposures, the experiment was carried out for 90 minutes with samples taken at t=0, 15, 30, 45, 60 and 90 minutes. At each time point, cells were lysed with 500μL Passive Lysis Buffer (PLB, Promega, Madison WI) and the effects of acute exposure were measured using the NanoGlo Dual-Luciferase^® Assay (Promega, Madison, WI). The maximal signal was observed for both bisphenols at 15 minutes. Therefore, for all subsequent acute exposures, samples were collected after 15 minutes.

Human 3A placental cells were used as the host for haplotype construct transfection. Cells were maintained in 75cm² flasks with Minimal Essential Medium with Earle’s Salts and L-Glutamine (Gibco Cat. 11095-080, Grand Island, NY) supplemented with 10% FBS in 5% CO₂ at 37°C. Cells were passaged at ≤85% confluency and sub-cultured at a 3:1 ratio. A solution of Trypsin-EDTA was used to detach the cells for sub-culture or transfer to 6-well plates for transfection. Transfections were performed between passages 6–8 with cells at low confluency (≤ 40%) with 2μL Fugene 6 (Promega, Madison, WI), 0.6μg promoter haplotype plasmid DNA, and 0.06μg of the constitutively active firefly luciferase plasmid pGL4.53 PGK (Promega, Madison, WI), which was used for in-well normalization of transfection efficiency. Transfection efficiency for 3A cells was determined using a green fluorescent protein plasmid (AcGFP1-C1) under these same transfection conditions. Cells were allowed to recover for 36–48h before harvest.