*4.1. Cell Cultures and Treatments*

The JEG-3 human choriocarcinoma cell line is widely used to study the molecular mechanisms underlying the proliferation and invasive potential of trophoblast cells [54]. The HEC-1A adenocarcinoma derived endometrial cell line is a suitable in vitro model for non-receptive and receptive endometrium [71]. JEG-3 trophoblast-like choriocarcinoma cells (ATCC, HTB-36) were cultured in EMEM medium (Lonza Ltd. Basel, Switzerland) supplemented with 10% fetal bovine serum (FBS, EuroClone S.p.A, Pero, Italy) 1% Non-essential amino acids (NEAA, Lonza Ltd., Basel, Switzerland), 1% sodium-pyruvate (Lonza Ltd., Basel, Switzerland) and 1% Penicillin-Streptomycin (P/S, Lonza Ltd., Basel, Switzerland). HEC-1A endometrial cells (ATCC, HT-112) were maintained in McCoy's 5A medium (Corning Inc., Corning, NY, USA) supplemented with 10% FBS and 1% P/S. For the experiments, the culture medium was supplemented with charcoal/dextran treated FBS (EuroClone S.p.A, Pero, Italy). For the monocultures, JEG-3 cells were seeded onto culture dishes (60 mm, Corning Inc., Corning, NY, USA) in the appropriate culture medium and were treated after a 24 h resting period. For the co-culture experiments, JEG-3 cells were seeded on culture dishes while HEC-1A cells were placed on Thermanox coverslips (Thermo Fisher Scientific Inc., Waltham, MA, USA). After 24 h, HEC-1A cells were added to JEG-3 cells by turning the endometrial cell holding coverslips upside down facing the trophoblast-like cells [42]. This way the cells were separated only by a thin layer of 1:1 mixture of supplemented EMEM:McCoy's 5A medium. Both mono- and co-cultures were treated for 6 h, 24 h and 48 h with 5, 10 and 20 ng/mL human recombinant fractalkine (Shenandoah Biotechnology Inc., Warwick, PA, USA). Untreated mono- and co-cultured cells were used as controls.
