Diff quik

Transwells (6.5 mm, 8 µm pore, Greiner BioOne) were coated with growth factor-reduced Matrigel (BD Biosciences, 400 μg/mL diluted in serum free RPMI-1640 medium) for 3 h. Medium was removed prior to plating cells. Rat TS cells were differentiated for 6 days, and then approximately 1.0 × 10⁴ cells were placed in the Transwells on top of the Matrigel. Transwells were then positioned in wells containing decidual conditioned medium supplemented with either PBS, normal goat IgG (1 μg/mL, AB-108-C, R&D Systems), or an OPN neutralizing antibody (1 μg/mL, AF808, R&D Systems), and incubated for 24 h at 37°C, 5% CO₂. After 24 h, excess cells and Matrigel were discarded from the top of the chamber using a cotton swab, and cells that invaded through to the underside of the Transwell were fixed in methanol and stained using Diff-Quik (GE Healthcare). Membranes were removed from the Transwell, placed on slides, and invaded cells counted under a microscope. For each condition, the total number of cells that invaded in 3 random fields of view per membrane was counted, and three independent membranes were used per experiment. Counts were averaged and normalized to the number of cells that invaded in the control condition to facilitate comparisons between experiments.

Transwells (6.5 mm, 8 µm pore, Greiner BioOne) were coated with growth factor-reduced Matrigel (BD Biosciences, 400 µg/ml diluted in serum free RPMI-1640 medium) for 3 h. Medium was removed prior to plating cells. Approximately 4.0 × 10⁴ HTR8 EVTs were placed on top of the Matrigel, and each chamber was then placed in normal culture medium and incubated for 24 h at 37 °C, 5% CO₂. After 24 h, excess cells and Matrigel were discarded from the top of the chamber using a cotton swab, and cells that invaded through to the underside of the transwell were fixed in methanol and stained using Diff-Quik (GE Healthcare). Membranes were placed on slides, and invaded cells counted under a microscope.