8 μm pore sized filters

A transwell migration assay was employed to investigate changes in the EC migration ability in response to various treatments as previously reported.¹⁵ In brief, 2 × 10⁴ (cells/well) ECs were plated into the apical chambers of transwell culture plates (24‐well; 8 μm pore‐sized filters; Corning) in 200 μL serum‐free medium, while the basolateral chamber was filled with 600 μL culture medium containing 20% FBS. Following an 8‐h incubation, the non‐migrated ECs were wiped off gently with a cotton swab, while the migrated cells on the bottom membrane surface were fixed with paraformaldehyde (4%) and stained with crystal violet solution (Heart) at room temperature for 30 minutes. Subsequently, the number of migrating cells was counted in five randomly selected microscopic fields. A scratch wound healing assay was also carried out to measure the EC migration ability as previously reported.¹⁶ More specifically, pre‐treated ECs were plated into 12‐well plates at a concentration of 2 × 10⁵ cells/well (Corning) and allowed to adhere overnight; then, a sterile 200 μL pipette tip was utilized to make a vertical scratch. To remove the dislodged cells, the wells were gently washed twice with PBS. Subsequently, the images of migrated cells were captured under a microscope at 0, 6 and 12 hours. ImageJ software was used to quantify the outcomes.

Transwell and scratch wound healing assays were used to evaluate cell migration in response to various CM-based incubations. For the transwell assay, ECs (2 × 10⁴ cells/well) suspended in 100 μL serum-free medium were seeded into the upper chamber of 24-well transwell culture plates (8-μm pore-sized filters; Corning), while 500 μL CM (H-CM, P-CM, H-GW, or P-GW), or the established control medium for H-CM and P-CM, was added to the lower chamber. After 8 h of incubation, nonmigrated cells on the upper surface of inserts were removed gently with a cotton swab, and then, the cells that migrated to the lower surface of membranes were fixed with 4% paraformaldehyde prior to staining with crystal violet (Heart) for 30 min. Images were captured, and the number of migrated cells was counted in five randomly selected fields per well. For the scratch wound healing assay, ECs (4 × 10⁵ cells/well) were seeded into 6-well plates (Corning). After the cells reached 90% confluence, one scratch was made in each well with a sterile 1 mL pipette tip, and then, the floating cells were removed by washing twice with PBS. After that, ECs were exposed to the various CMs. Scratch images were obtained at baseline (0 h) and at different observation time points (12 h; 24 h), and the outcomes were analyzed with ImageJ software.