2 well silicone culture insert

The cells of 2.8 × 10⁴ in 70 μL of the medium were cultured on 2-well silicone culture insert (ibidi, Bavaria, Germany) settled in 24-well plate for 24 h. The insert was removed, and the cells were washed with PBS at 37 °C. Then, cell migration was initiated in the medium containing with 30 μM JPH203 and/or 5 μM CX-4945 with final DMSO concentration of 0.8%. After 24 h cultivation for migration, cells were washed with PBS, followed by staining with 0.25% crystal violet in 20% methanol for 10 min. After washed with PBS five times, the migrated cells were subjected to image acquisition using an inverted microscope CMi1 (Leica Microsystems, Wetzlar, Germany). The number of migrated cells with inhibitor treatment was counted by using ImageJ software with Cell Counter plugin (NIH), and expressed as % of the control without inhibitor treatment.

Cells were plated in an Ibidi 2-well silicone culture insert (Ibidi, catalog # 81176) adhered to a TC-treated 35-mm glass bottom dish and grown to confluence. At confluence, culture inserts were removed to create a “scratch.” Dishes were washed with DPBS to remove unattached cells, and the media was changed. Cells were incubated in control (normoxia) or hypoxia (1% O₂) conditions for 6 h. Live cells were imaged using a Zeiss Axio Observer 7 inverted microscope fitted with a 10 × objective at 1, 2, 4, and 6 h. Cell migration was calculated as the percent difference in the cell-free area in hypoxic dishes compared to control at each time point using the ScratchAssayAnalyzer function in the MiToBo FIJI plugin.

HUVECs were seeded at 2.8 × 10⁴ cells in 70 μL of EGM-2 medium/well in 2-well silicone culture insert (ibidi GmbH) settled in 24-well plate, and incubated for 18 h. After starvation for serum and growth factors in EBM-2B medium (EBM-2 medium supplemented with 0.1% BSA) for 6 h, cell migration was initiated by removing the inserts and adding 1 mL/well of EBM-2B medium containing 10 ng/mL VEGF-A. DIC images were acquired immediately after removing the inserts to locate the initial edges of cell-free gaps. Cells were incubated for 12 h for migration, fixed by 4% paraformaldehyde (PFA), stained with crystal violet, and subjected to image acquisition. Bright field images were acquired using an inverted microscope (DMi1, Leica Microsystems). The number of migrated cells were counted by using Cell Counter plugin for ImageJ software (NIH).