2 well silicone insert

DLD‐1 wt cells were plated at a density of 5 × 10⁴ cells/well in 2‐well silicone inserts (ibidi GmbH, Gräfelfing, Germany) and incubated at 37 °C for 24 h. Then, inserts were removed leaving a 500‐µm gap without any cells. Dishes were cultured for 24 h in McCoy’s 5A medium (Invitrogen), supplemented with 10% FBS (Invitrogen), and in the presence, or absence, of TIMP‐1 (5 µg·mL⁻¹), imatinib mesylate (Sigma‐Aldrich), or both, and imaged for gap size measurement. Three nonoverlapping representative images from each of the gapped areas were acquired to estimate the relative migration of cells. Each condition was examined in triplicate experiments.

Briefly, cells were grown until confluent in 2‐well silicone inserts (Ibidi®, Germany) placed in 12‐well tissue culture dishes. The cell culture inserts were removed after 1 day. Afterward, the plates were washed with PBS and incubated at 37°C in fresh RPMI‐1640 medium (Gibco BRL, Invitrogen, Paisley, UK) supplemented with 10% fetal bovine serum (Eurobio) and 1% penicillin–streptomycin antibiotics (Gibco, Invitrogen), either naive or in the presence of vehicle (DMSO) or CH‐223191 (5 μM). The wound was photographed with an inverted microscope at 5× magnification using an Axio Vert.A1 inverted microscope (Carl Zeiss). Wound closure was determined by measuring the distance between the edges of the wound at time 0 and 15 h using ImageJ (Fiji). Quantification of the distance migrated by the cells was performed as follows: $$\mathrm{D}=\left(\text{size of the wound}\mathrm{at}t=0\mathrm{h}-\text{size of the wound}\mathrm{at}t=15\mathrm{h}\right).$$

Wound healing was assayed using 2-well silicone inserts (ibidi GmbH, Gräfelfing, Germany) placed into a 24-well plate. After 24 h of starvation, 5 × 10⁴ cells/well were plated with 70 µL 10% FCS/DMEM. The cell culture inserts were removed after 24 h leaving a defined cell-free gap of 500 μm. At this time point (0 h), 1.5 mL of 10% FCS/DMEM were added into each well of the 24-well plate and images were taken at 10× magnification. The collagen matrices were then placed over the gap and co-cultured with the cells for 24 h. After careful removal of the matrices, images for the 24 h-time point were captured on a Leica DM IL LED microscope equipped with Leica DFC420 C camera. The cell-free area at 0 (T0) and 24 (T24) h was determined by using ImageJ as described [30 (link)]. The percentage of wound closure was calculated using the following formula: [(T0–T24)/T0] × 100. Data represent means ± SD from three independent experiments performed with three different cell donors per cell type, each in triplicates.

The rate of wound closure was observed using 2-well silicone inserts (ibidi, Gräfelfing, Germany). The HLEC suspension was adjusted to a cell concentration of 5 × 10⁵ cells/ml. Seventy microlitres of cell suspension was added to each well. The cells were cultured overnight at 37 °C with 5% CO₂. The 2-well inserts were gently removed with sterile forceps and washed twice with PBS. Cell medium with or without TGF-β2 was added to each well. Photographs were taken every 12 h for a total of 48 h using a Cytation™ 5 Cell Imaging Multi-Mode Reader (BioTek, Vermont, USA).

Wound healing was assayed using 2-well silicone inserts (ibidi GmbH, Gräfelfing, Germany) placed into a 12-well plate. A total of 5 × 104 cells/well were plated with 70 µL 10% FBS medium. The cell culture inserts were removed after 24 h, leaving a defined cell-free gap of 500 μm. A total of 0.5 mL of 10% FBS medium was added into each well of the 12-well plate at 0 h and images were taken at 40× magnification. The cell numbers at 16 h were determined by using image analysis software (FIJI, ImageJ V1.52p (National Institutes of Health, Bethesda, MD, USA). Data represent means ± SD from three independent experiments performed.

After 72 h treatment with 0.1 µg/ml doxycycline, 7 × 10⁵ HCC1954-luc shNEG and HCC1954-luc shEDI3 cells were re-plated into 2 well silicone inserts (Ibidi) and allowed to attach overnight. Culture inserts were removed using sterile tweezers, leaving a clean gap between the two cell monolayers. Directly upon removal of the insert, as well as on subsequent days, four photos per well were taken at the same position until the gap in the non-induced shNEG cells was almost fully closed. Alternatively, migration was investigated using scratch assays as described previously [11 (link)]. The size of the gaps was determined using the “wound healing tool” of the ImageJ software. The average percentage of “wound” closure was calculated from all four photographed positions and compared to the non-induced shNEG cells or cells transfected with scrambled siRNA (siNEG).

The wound-healing assay was performed using 2 well silicone inserts with a 500 µm cell-free gap (Ibidi, Gräfelfing, Germany). HaCaT cells were seeded in 12-well plates at a density of 9 × 10⁵ cells/mL in DMEM growth medium containing 10% FBS in inserts. According to manufacturer’s protocol, 70 µL was applied in every well. To reach confluency, cells were incubated overnight at 37 °C in 5% CO₂. Thereafter, inserts were removed with sterile tweezers. First, 990 µL DMEM growth medium was applied to the cells, then 10 µL bark extracts diluted in H₂O was added to final concentrations of 25, 50 and 100 µg/mL. Cells were imaged immediately, and after 24 h and 48 h. Gaps were analyzed using TScratch.