Mab1880

Non-labouring term human placentae were donated with informed consent by women delivering uncomplicated singleton pregnancies via elective caesarean section at the John Hunter Hospital, Newcastle, Australia. Exclusion criteria are as previously described [10 (link)].
Primary trophoblast isolations were performed as previously described by Kaitu’u-lino et al. within 30 min of delivery and included performing negative selection using a CD9 antibody (MAB1880, R&D Systems, MN, USA), ensuring a pure population of trophoblast cells [11 (link)]. Cells were plated as previously described in Morosin et al. [10 (link)]. Briefly, 12 or 24-well plates with coverslips inserted were coated with fibronectin (10 μg/mL), prior to trophoblast cell seeding at 800,000 cells/well. Trophoblasts were maintained in 1× high-glucose Dulbecco’s Modified Eagles Medium (DMEM-HG; Hyclone, UT, USA) supplemented with 10% heat inactivated fetal bovine serum (FBS; Bovogen Biologicals, Vic, Australia), 2 mM L-glutamine (Gibco, CA, USA) and 1% antibiotic-antimycotic (Gibco) in 5% CO₂ in room air at 37 °C. Primary trophoblast cell cultures were probed with Cytokeratin 7 (CK7) using immunocytochemistry (ICC) to assess the purity of the cell population. Assessment of CK7 levels indicated that 93% of the cells were trophoblasts (N = 5 placentae; data not shown).