Proliferation and Viability of hfBECs

Proliferation and viability of early and mid-pregnancy hfBECs were determined by XTT dye-reduction assay and trypan blue cell exclusion, respectively, as previously described [37 (link),38 (link)]. For the cell proliferation assay, 5000 cells/well were plated into 96-well plates and maintained in culture for 3 days. On the day of assay at 24, 48 and 72 h, 50 μL of XTT (5 mg/mL; X6493, Invitrogen, Burlington, ON, Canada) and 0.4 μL of phenazine methosulfate (1 mg/mL; PSM; Sigma) were added to each well and the cells were incubated (37 °C for 3 h). Absorbance was measured at 450 nm. For cell viability assessment, cells (15,000/well) were seeded into 6-well plates. Trypan blue was added at 24, 48 and 72 h, and the ratio of cells containing dye vs those that did not was calculated.