Dmi4000 automated inverted microscope

Wound-healing assays were performed by creating identical wound areas into the cell monolayer using Ibidi culture-inserts (Ibidi GmbH, Cat. no. 80209, Munich, Germany). U2OS cells were seeded in complete culture medium at a density of 3 × 10⁴ cells on each side of the Ibidi culture-insert, into a 24-well plate. After attachment, cells were treated for 24 h inhibitors (or vehicle in the controls); then the culture-insert was detached in order to form a cell-free gap into the cell monolayer. Each well was rinsed once with phosphate-buffered saline (PBS) to remove cell debris and immediately refilled with fresh medium, with new addition of the inhibitors (samples with “maintained” treatment) or without (sample with “removed” treatment). Cells were allowed to migrate for further 48 h. The wound images were captured at time zero (t = 0 h), t = 24 h, and t = 48 h using a Leica DMI4000 automated inverted microscope equipped with a Leica DFC300 FX camera.

U2OS cells were plated at 50% confluence, allowed to adhere to the plate for 16 h, and then treated with inhibitors or vehicle for 24 h. After treatment, cells were washed, detached, counted, and seeded at 1000 cells/well in 200 μL of culture medium, in a 96-well plate with round bottom wells precoated with 50 μL of 1% agar in culture medium [17 (link)]. Images were taken from each well 24 h and 96 h later, by means of a Leica DMI4000 automated inverted microscope equipped with a Leica DFC300 FX camera.

Wound-healing assays were performed by creating identical wound areas into the cell monolayer using Ibidi culture-inserts (Ibidi GmbH, Cat. no. 80209). Cells were seeded in triplicate in complete culture medium on each side of the Ibidi culture-insert, into a 24-well plate, at proper cell densities (1.5 × 10⁶ SK-N-BE cells/mL and 5.5 × 10⁴ U2OS cells/mL). After 24 h, cells reached confluence, and the culture-inserts were removed in order to form a cell-free gap into the cell monolayer. Each well was washed once with PBS to remove cell debris and immediately refilled with fresh D-MEM medium supplemented with 5% (v/v) FBS and 2mM L-glutamine. The wound images were captured at this time (t = 0) and at the indicated time points (24–48 h for SK-N-BE, 2–4–8 h for U2OS) using a Leica DMI4000 automated inverted microscope equipped with a Leica DFC300 FX camera (2.5× objective, magnification-changer 1.6). Images of in vitro scratch wound healing assays were analyzed with a plugin of ImageJ software (1.52t version) adapted from [28 (link)] and relative cell migration of each cell line was measured.