Transwell invasion assay

The involvement of human subjects in this study was approved by the Institutional Review Board of The University of Hong Kong/Hospital Authority Hong Kong West Cluster. Human placental tissue was collected from the termination of pregnancy in the first 12 weeks of gestation performed in the Department of Obstetrics and Gynaecology, The University of Hong Kong. All samples were processed immediately after collection. The primary trophoblast was isolated by our published protocols [10 , 11 (link)]. The trophoblast invasion was quantified by a transwell invasion assay (Corning, NY). In brief, primary human trophoblasts (5 × 10⁵) in serum-free DMEM with/without supplementation of 10 nM ADM or 100 μM GSNO were allowed to invade through a basement membrane for 24 h. The invaded cells on the membrane were stained with 0.1% crystal violet and quantified by measuring the absorbance at 595 nm after dissolving the crystal violet dye from the membrane by 10% acetic acid.

A cell suspension containing 5×10⁵ cells/ml was prepared in serum-free media for the Transwell invasion assay (Corning Inc., Corning, NY, USA). Then, 500 μl of DMEM supplemented with 10% FBS was added into the lower chamber and 300 μl of the cell suspension was added to the upper chamber. The cells were then incubated at 37°C with 5 % CO₂ for 24 h. Following incubation, the cells on the upper surface were removed using a cotton-tipped swab, while the cells on the lower surface were stained for 30 min. Under the microscope, the cell number was counted in at least five randomly selected fields.

According to the manufacturer’s instructions, 8-μm pore filters were used for Transwell invasion assays (Corning, Bedford, USA) to determine the invasion. In brief, HUVECs (3×10⁴ cells) treated with miR-199a-5p mimics or inhibitor for 24 hours were washed and resuspended in RPMI 1640 medium supplemented with 0.1% BSA. The cells were then placed on the upper part of a 24-well Transwell insert coated with Matrigel^® (Corning, Bedford, US), while the lower chamber contained endothelial cell medium (ScienCell, USA). After 6 hours, the filter was removed, fixed and stained with crystal violet staining solution (0.1%, Solarbio Life Sciences, China).

The 24-well culture plate, including transwell upper chamber inserts, was used for transwell invasion assays (Corning, New York, USA). First, 1 × 104 cells or cells transfected with OE-RNA and/or control vectors were mixed with 100 ml serum-free RPMI-1640 or DMEM with different concentrations of ibuprofen (C643 at 0, 0.5, 1, and 1.5 mM, and OCUT-2C at 0, 1, 2, and 3 mM) added to the upper chamber. Then, 600 ml of medium containing 10% FBS with the same concentrations of ibuprofen was added to the lower chamber. After culture at 5% CO₂ at 37 °C for 48 h, the chamber was removed from the plates and fixed with 4% paraformaldehyde for 30 min. The cells that traversed through the membrane pores were stained with crystal violet. Ultimately, the number of cells passing from the upper chamber to the lower chamber was observed through an inverted microscope (Olympus, Tokyo, Japan).