Glomax multimode reader

The viability and cytotoxicity of HUVECs were analyzed using a RealTime-Glo™ MT cell viability assay kit (Promega, Madison, WI, USA), according to the manufacturer’s instructions. Briefly, HUVECs were plated into 96-well white cell culture plates at a density of 5 × 10³ cells/well. After 24 h of incubation, the pro-substrate and luciferase were added at the same time as that of the hormones to continuously monitor the viability of the HUVECs in real-time. Luminescence intensity at the desired time points was measured using a Glo-Max™ multimode reader (Promega, Madison, WI, USA).

The devices were cultured initially for 14 days in either complete growth medium, or complete EBM-2 medium supplemented with oestradiol (1 nM) until both cell monolayers reached 80% confluence. The complete growth medium for the stromal chamber was then supplemented with MPA (0.5 nM) and media was collected and changed daily (300 µL) for an additional 14 days from both inlet and outlet. Collected effluents were then analysed by measuring prolactin production, a marker of decidualization, using an enzyme-linked immunosorbent assay (ELISA Duoset, R&D systems). The ELISA was performed according to the manufacturer’s instructions using 50 µL samples. The plate was read using an absorbance microplate reader (GloMax^® Multimode Readers, Promega). The results are representative of three different experiments. Due to the patient-related variability among experiments, we normalized prolactin concentration to the minimal mean prolactin production at day 2. Statistical analysis was performed by a 2 way ANOVA using a Bonferroni correction. Statistical significance was calculated as p < 0.05.

A 3 mL syringe was filled with a 2.5 mg/mL solution of FITC-dextran (150 kDa MW, Sigma Aldrich, USA) and mounted on a syringe pump. The syringe was connected to the top chamber with microbore tubing, while 200 µL of 1X PBS were added to the outlet reservoir and to the inlet and outlet of the bottom chamber. Perfusion was run at 2.5 µL/min. 100 µL samples were collected from each of the reservoirs and replaced with 100 µL of PBS at 1, 2 and 3 h intervals. Fluorescence intensity in the collected effluent was measured using a fluorescence microplate reader (GloMax^® Multimode Readers, Promega) at 470 nm excitation and 520–550 nm emission range (details in Supplemental Material).