Cells were plated in plastic flasks (75 cm2, Falcon, Franklin Lakes, NJ), maintained at 37°C in a humidified atmosphere (5% CO2 in air), and grown to confluence. The stromal cells were passed by trypsinization and plated in culture dishes (100-mm diameter) and were allowed to replicate to confluence. Immunocytochemical analysis of endometrial cells was conducted after the first passage.
Factor VIII, cytokeratin, 3C10, and vimentin were used as markers of endothelial cells, epithelial cells, macrophages, and stromal cells, respectively. Ninety-seven percent of the cells were endometrial stromal cells. Epithelial cells and macrophages accounted for ~3% and 0.2% of the cells; endothelial cells were absent. The 70%-80% confluent monolayers were maintained in serum-free media for 24 h and subsequently treated with 17p-estradiol (E2; 1 X 10~8 M; Sigma) or progesterone (1 X 10~6 M; Sigma) for 24 h. Steroids were dissolved in ethanol and diluted to a final concentration of less than 0.001%. Control cells were treated with an equal amount of ethanol.
Ishikawa cells, a well-differentiated endometrial adenocarcinoma cell line, were cultured in phenol red-free Eagles minimum essential medium containing 10% (v/v) charcoal-stripped FBS and supplemented with penicillin (100 |xg/ml), glutamine (2 mM), and sodium pyruvate (1 mM). Estrogen and progesterone receptor status were verified by ELISA according to the manufacturer’s instruction (Abbot Laboratories, Weisbaden, Germany). The 70%-80% confluent monolayers were maintained in serum-free media for 24 h and subsequently treated with 17^-estradiol (E2; 1 X 10~8 M; Sigma) or progesterone (1 X 10~6 M; Sigma) for 24 h.