BeWo: Cells destined for transfection using an electroporation-based system - nucleofection, were grown in T75 flasks until 70–80% confluent, then trypsinised and counted. 1 Ü? 106 cells/condition were resuspended in cell line solution L or cell line solution V and transfected following the manufacturers' instructions using programs A-020, T-020, X-001 and X-005 (Amaxa Biosystems, Germany). Following transfection, cells were plated at approximately 7 Ü? 10^4/well for 24-well plates and approximately 3 Ü? 10^5/well for 6 well plates.
Primary cytotrophoblast cells:
Cells intended for transfection by nucleofection were centrifuged at 1000 g for 5 min immediately after isolation and then resuspended in the nucleofection buffer (100 μl/2 Ü? 106 cells) provided in the basic primary mammalian epithelial cell nucleofector kit (Amaxa Biosystems, Germany). 2 Ü? 10^6 cells/condition were transfected using programme Nucleofector programmes S-005, T-020, T-023 or U-017 and then plated onto 12 well plates at 1–2 Ü? 10^6 cells/well in 1.5 ml culture medium (DMEM/Ham's F12 (1:1, v/v) containing 10% fetal calf serum and antibiotics).
1st trimester human placenta explants:
In order to carry out transfection by nucleofection, 2–3 pieces of placental tissue were placed in 100 μl basic primary epithelial cell nucleofector buffer containing siRNA or vehicle. Using a wide-ended 1.5 ml Pasteur pipette, tissue and buffer were transferred to a Nucleofector cuvette and then exposed to programs X005, X001, U006, U007 and U017 before removal using pipettes supplied with the Nucleofector kit.