Falcon 353 047

The invasion kit was purchased from BD Falcon 353,047 (Franklin Lakes, NJ, USA). The invasion assay was performed according to the manufacturer's instructions in normal cell culture serum. Cells were seeded in Transwell chambers and fixed with 3.7% formalin for 2 min, washed twice with PBS, transferred to 100% methanol for 20 min, washed twice again, and finally stained by Giemsa (1:20 diluted with PBS) at room temperature for 15 min in the dark. After staining, the cells were washed twice with PBS, and non-invaded cells were scraped off with a cotton swab (PBS wetted) four times. Images were captured under an Olympus DP71, and the number of cells was calculated using Image J software as described previously [18] (link), [23] (link).

This work followed the tenets of the Declaration of Helsinki involving human subjects and received approval from our institutional review board. Human AM tissue (ie, the innermost layer of the placental membrane) was obtained with informed consent at the time of elective cesarean section from mothers when human immunodeficiency virus, syphilis, and hepatitis B and C had been excluded by serological tests. Separation of the AM was performed by cutting the tissue samples approximately 2 cm from the placental disc. The average thickness of the AM samples was about 100 μm. EDC, l-lysine, ninhydrin reagent, MMP-1 (EC 3.4.24.7), and MMP-9 (EC 3.4.24.35) were obtained from Sigma-Aldrich (St Louis, MO, USA). N-hydroxysuccinimide (NHS) was supplied by Acros Organics (Geel, Belgium). Balanced salt solution (BSS, pH 7.4) was purchased from Alcon Laboratories (Fort Worth, TX, USA). Dulbecco’s Modified Eagle’s Medium and TRIzol reagent were purchased from Gibco-BRL (Grand Island, NY, USA). The 24-well tissue culture polystyrene plates (Falcon 353047) and cell culture inserts (Falcon 3095) were purchased from Becton Dickinson Labware (Franklin Lakes, NJ, USA). All other chemicals were of reagent grade and were used as received without further purification.