Elh pp14

For the differentiation of both stromal-only and EO-only scaffolds, 10 nM β-oestradiol (E2, Sigma) was added 2 days post-cell seeding. For stromal cells only, at days 4 and 6, medium was replaced with the following conditions: 10 nM E2 + 1 μM progesterone (P4, Sigma) + 1 μM 8-bromoadenosine 3′,5′-cyclic monophosphate (cAMP, Sigma). For EO-only scaffolds, 20 ng ml⁻¹ prolactin (PRL, Peprotech, UK) was added in addition at days 4 and 6 to mimic signals from decidualized stromal cells. Supernatant was collected at days 2, 4, 6 and 8 and frozen at −20°C for storage before downstream analysis.
Enzyme-linked immunosorbent assays (ELISAs) were used to measure protein concentrations of key markers of stromal decidualization (prolactin, a hormone secreted by decidual stromal cells) and epithelial differentiation (glycodelin, one of the principal components of the endometrial glands) in the supernatant. ELISAs for prolactin (DY682, R&Dsystems, USA) and glycodelin (ELH-PP14, Raybiotech, USA) were measured from stromal-only and EO-only scaffolds, respectively. A volume of 100 μl of supernatant was used with each sample measured in duplicate, as per the manufacturer's instructions.

At the end of each hormonal treatment experiment, culture medium was pooled from four wells per condition and centrifuged at 12,000 RPM for 5 min. Supernatants were transferred to a fresh tube and stored at −80 °C until analysis. Human PP14/glycodelin A ELISA (RayBiotech Inc., ELH-PP14) was performed using 100 μl supernatant in duplicate alongside a series of diluted human glycodelin standard according to the manufacturer’s instructions. The concentration of glycodelin in the supernatants was calculated from the line formula of the standard plots.