Cellsens imaging software 2

Twenty-four hours after transfection or plating, cells were seeded in 2-well silicone inserts with a defined cell-free gap of 500 µm (ibidi^®, DE, cat# 80209) at high confluence in 6-well plates. The following day, the inserts were removed, cells washed twice with sterile PBS and complete growth medium supplied. Wound closure was followed over time, at the indicated times. Migrating cells were imaged with an IX71 inverted microscope (Olympus, Tokyo, JA) provided with a CellSens Imaging Software 2 (Olympus, Tokyo, JA; RRID:SCR_016238). The area of wound closure was calculated using the ImageJ version 1.52.n (University of Wisconsin, WI, USA; RRID:SCR_003070) with respect to the initial area (T₀) and expressed as percentage of wound healing at the time-points indicated.

Twenty-four hours after re-expression of the plasmid constructs, 10,000 cells were seeded in 12-well plates. After 24 h and 48 h, cells were washed with PBS, fixed-and-stained with a 0.025% (w/v) Crystal violet (Sigma-Aldrich; cat# C6158) solution in 20% (v/v) MeOH on ice for 15 min. After washing with ddH₂O, plates were air-dried and pictures taken with an IX71 inverted microscope (Olympus) provided with a CellSens Imaging Software 2 (Olympus). For quantitation, Crystal violet was eluted with 100% MeOH and absorbance measured at 595 nm by a VICTOR Multilabel Plate Reader (PerkinElmer). Data are expressed as fold change with respect to absorbance of control sample (WT at 24 h).

Ten-thousand cells from monoculture were seeded onto Nunc™ Polycarbonate Cell Culture Inserts in Multi-Well Plates (ThermoFisher Scientific, MA, USA; cat# 140629) in FBS-free growth medium 24 h after transfection or at the moment of seeding (ESTDAB and primary cells). Growth medium supplemented with 10% FBS was used in the bottom chamber as attractant. Cells were incubated at 37 °C for 24 h and then fixed and stained with a 0.05% (w/v) Crystal Violet solution in 20% MeOH. Cells on the top section of the insert were gently removed with a cotton swab. Four different fields were imaged for each sample with an IX71 inverted microscope (Olympus, Tokyo, JA) provided with a CellSens Imaging Software 2 (Olympus, Tokyo, JA; RRID:SCR_016238) and counted with ImageJ version 1.52.n (University of Wisconsin, WI, USA; RRID:SCR_003070). Data are showed as average count.