Transwell insert

Cell migration and invasion were assessed using Transwell assays (46 (link)). The TE-1 and Eca-109 cells suspended in serum-free medium were plated in the upper chamber of Transwell inserts (8 µm-pore size, EMD Millipore) with Matrigel (for invasion assay) or without Matrigel (for migration assay). Medium containing 20% fetal bovine serum was plated into the lower chamber. Following incubation for 48 h at 37°C, cells remaining on the upper chamber were wiped away using a cotton swab. Cells migrating into the lower surfaces of the Transwell inserts were fixed in methanol and stained with 0.5% crystal violet (Beyotime Institute of Biotechnology) at room temperature for 30 min. The results were detected using a Zeiss photomicroscope and counted based on at least 10 random visual fields.

PC-3^Luc cells were seeded (2.0×10⁴ cells/well) onto 6-well culture plates. M-CSF expanded bone marrow–derived macrophages were loaded (1:3) into top cell culture 0.4-μm Transwell inserts (EMD Millipore) alone or with UV-induced apoptotic PC-3^Luc cells on day 0 and again on day 4. To measure tumor cell growth, Transwell inserts were removed, and tumor cell growth was measured using bioluminescence.

Following the indicated treatments, the culture medium was removed and replaced with EBM-2 without any supplement. After 12 h, EPC migration was evaluated by using a Transwell migration assay. Briefly, 3×10⁴ cells were suspended in 100 µl of EBM-2 supplemented with 0.1% BSA and placed in the upper chamber of 8.0-µm pore size Transwell inserts (Merck Millipore). VEGF in serum-free EBM-2 was placed in the lower compartment of the chamber. After incubation for 24 h at 37°C in 5% CO₂, the cells that had not migrated were removed from the upper surface of the filters using cotton swabs and those that had migrated to the lower surface of the filters were fixed in 4% paraformaldehyde and stained with 4′,6-diamidino-2-phenylindole (Roche Applied Science, Indianapolis, IN, USA). Cells that had migrated into the lower chamber were counted manually in five random microscopic fields at a magnification, ×100.

The assay was carried out with transwell inserts (EMD Millipore, Billerica, MA, Germany) coated with or without Matrigel (BD Biosciences San Jose, CA). Briefly, 2×10⁴ cells and 300 μL serum-free DMEM were placed in the upper chamber (pore size, 8 µm; Millipore, Germany), and 1000 μL DMEM containing 10% FBS was added to the lower chamber. After 48 hrs, the cells that had migrated to or invaded the lower surface of the filter were fixed and stained, and the mean values for the cell counts in five random fields of vision under a microscope (×200) were calculated.

CMVECs (5×10⁴) were seeded into the upper chambers of the Transwell inserts (pore size, 8 µm; EMD Millipore) with FBS-free medium. DMEM supplemented with 20% FBS was plated into the lower chambers. Following treatment with H/R and cell culture for 48 h, migratory cells were fixed in 4% paraformaldehyde at room temperature for 30 min and stained with 0.1% crystal violet (Beyotime Institute of Biotechnology) at room temperature for 15 min. Following washing twice with PBS, migratory cells were observed and imaged at ×200 magnification using an inverted optical light microscope (Olympus Corporation) and the number of cells was quantified for analysis.