Eclipse ti s phase contrast microscope

Cells (3.5 × 10⁴) were seeded and cultured into the inner wells of cell culture inserts (ibidi, Germany) and placed in a Petri dish. Once attached to the substratum, the inserts were removed from the surface leaving a 500 μm cell-free wound. To evaluate basal migration, the wounded areas were photographed at 0 and 48h with a CoolSNAP camera (Photometrics) coupled to an ECLIPSE Ti-S phase contrast microscope (Nikon, Japan) and processed using MetaMorph 7.6.5.0 image analysis software (Molecular Device, CA). Wound healing was measured as follows: [1- (empty area at 48h/ empty area at 0h)] x100.

To evaluate cell migration, wound-healing assays were performed according to previously reported protocols [49 (link), 50 (link)]. Briefly, cells were seeded in fibronectin-coated 6-well plates, serum-starved overnight in media containing 1% FBS, and pre-treated as indicated for two days until reaching the appropriate confluence on the day of the experiment. The monolayers were then lightly scratched with a 200 μl or 1 ml pipette tip. Floating cells were washed off with PBS, and the remaining cells were cultured in serum-free media. Images of the same fields for each condition were visualized with a Nikon Eclipse Ti-S phase-contrast microscope with ×100 magnification at two preselected time points (0 h and 24 h). The wounded area was defined in each image by positioning green lines corresponding to the original scratch. The number of cells that migrated into the wounded area at 24 h was visually counted. The results (number of migrated cells) were presented as the mean ± SD of three independent experiments performed in triplicate. For cell invasion assessment, Transwell chamber assays were performed according to a protocol that was thoroughly described previously in one of our studies [24 (link)].