Transwell chambers in 24 well plates

Transwell chambers in 24-well plates were purchased from Corning (Corning, NY, USA). Two hundred microliters of serum-free medium containing 1 × 10⁵ cells was added to the upper chambers. Then, 600 µL of medium supplemented with 20% FBS was added to the lower chambers as an attractant. Cells were cultured for 24 h and washed with PBS. Cells remaining on the upper surface of the chamber membranes were wiped off with a cotton swab. Cells that migrated to the bottom surface of the chamber membranes were fixed with 4% formaldehyde and stained with 0.5% crystal violet. Cells were counted in five random photographed fields.

Cells from the three trophoblast lines (5 × 10⁵ cells) were seeded in Transwell chambers in 24-well plates (Corning, NY, USA). Fifty microliters of a 1:10 dilution of Matrigel™ was used to cover the pore membranes (8 μm) in twenty-four-well plates. The trophoblasts were cultured on the membranes for 12 h. One of two types of medium was added to the lower chamber: CV-MSC CM supplemented with 10% FBS or normal culture medium supplemented with 10% FBS (control). Transwell assays were performed while culturing the trophoblasts without FBS for 24 h. Next, 4% paraformaldehyde was used to fix all cells. Trophoblasts that penetrated through the membranes were analysed. The cells that migrated through the membrane were counted in high-magnification fields, and every experiment was performed three times.