Dp2 bsw software

The Three-Dimensional (3D) vascular invasion assay was performed in transwell chambers using 24-well fitted inserts with 8-μm pore size. A mixture of 8 × 10⁵ EaHy-926 endothelial cells/well in 15 μL of Matrigel (Sigma; 1:1 v/v in DMEM/F12) was plated on the upper chamber, and DMEM/F12 was added in both compartments after polymerization. The cells were kept until ∼100% confluence was achieved. Afterward, HTR-8/SVneo cells were stained with 1 μM of carboxyfluorescein succinimidyl ester (CFSE, Thermo Fisher Scientific) and kept in the dark for 10 min. The CFSE-stained HTR-8/SVneo cells were placed at 2 × 10⁵ cells/well on top of the previous mixture of Matrigel and EaHy-926 cells, and supplemented DMEM/F12 was added in both compartments (5% FBS in an upper compartment with 10-μM uvaol, and 20% FBS in the lower compartment). After 1 h, GBS was added to the upper compartment. After 48 h, non-invading cells from the upper compartment were removed with a cotton swab, supernatants were collected, and the membranes were fixed with ice-cold methanol. Nuclei were stained with DAP-I, and the cut membranes were mounted with PBS/glycerol (1:9, v/v) under glass slides. The results were visualized with fluorescence microscope Nikon DS-Ri1 (Nikon, Japan), and images were acquired at 400 × magnification using the DP2-BSW software (Nikon).

HTR-8/SVneo cells were plated at 3 × 10⁵ cells, and, after 24 h, uvaol was added 1 h before GBS incubation and maintained for 24 h. The cells were then fixed with 4% paraformaldehyde in PBS and permeabilized with.1% Triton X-100 in PBS (v/v). Phalloidin-fluorescein (FITC)-conjugated staining (1:100; Abcam, Cambridge, United Kingdom) was added for 1 h, and nuclei were stained with DAP-I. Cells were mounted with PBS/glycerol (1:9, v/v) under glass slides, and the results were visualized with fluorescence microscope Nikon DS-Ri1 (Nikon). Images were acquired using the DP2-BSW software (Nikon).

After the previously described treatments, MitoSOX red (Thermo Fisher Scientific, Waltham, MA, United States) staining was incubated in living cells, following the conditions of the manufacturer. After 30 min, cells were fixed with 4% paraformaldehyde in PBS (v/v), washed with PBS, stained with 4’,6-diamidino-2-phenylindole (DAP-I), and mounted with PBS/glycerol (1:9, v/v) under glass slides. The results were visualized with a fluorescence microscope Nikon DS-Ri1 (Nikon). Images were acquired using the DP2-BSW software (Nikon). The percentage of cells with Mitochondrial Reactive Oxygen Species (mtROS) was determined by [(number of cells positively stained by MitoSOX/total number of cells) × 100].