Silicone culture insert

To study the effects of treatment on cell migration, a monolayer wound healing assay was performed. A silicone culture insert (Ibidi GmbH, Martinsried, Germany) was inserted into each well of a 4-well μ-Slide (Ibidi GmbH), and 70 μL of H1_DL2 cells (concentration: 9 × 10⁵ cells/mL in cell culture medium) were added into each half of the culture inserts. The cells were allowed to grow to confluence for 24 h, before the culture insert was removed, resulting in a wound (a cell free gap) with a width of 500 μm within the culture. Drugs were then added into the wells (5 μM vemurafenib, 5 μM temsirolimus or 5 μM vemurafenib + 5 μM temsirolimus), and cell migration into the wound was studied by time lapse microscopy for 60 h, using a Nikon TE2000 inverted microscope (Nikon Instruments Inc., Melville, NY, USA) equipped with an incubator holding 37 °C, 100% humidity and 5% CO₂. Pictures were then processed and analyzed with Image-Pro Plus (MediaCybernetics, Warrendale, PA, USA). The experiments were performed in triplicate.

Each cell population was seeded in two wells of silicone culture inserts (Ibidi GmbH, Gräfelfing, Germany) that were placed onto a single glass of the observation chamber (measuring 60 × 35 × 0.2 mm) at a density of 2.5 × 10³/cm² in complete culture medium. After 24 h, the medium was replaced with serum-free DMEM HG medium that contained 0.1% BSA. After another 24 h, TGF-β was added (human natural, Corning Incorporated, Corning, NY, USA; (356040)) at a final concentration of 5 ng/mL. Control cells in the neighbouring compartment remained untreated. After 5 days of incubation, dcEF application and time-lapse imaging were performed using the method described earlier. Cells that presented a phenotype of myofibroblasts (i.e., well-spread cells with significantly larger areas) were selected for quantitative analysis.

Between 5000 and 6000 NHLF cells expressing α-SMA were plated in 12-well plates with silicone culture inserts (Ibidi, GmbH, Planegg, Germany) for 48 hours with either 5 μg/mL type IV collagen protein or 0.5 M acetic acid as a control. Inserts were removed, fresh treatment added, and cell migration monitored during 24 hours. Images of the different time points captured on a Nikon DS-QiMc camera using NIS-Elements BR 3.0. Quantification of cell invasion into the insert space was performed and compared using ImageJ software. Experiments were conducted three times (n = 3) in triplicate. Gap cell invasion was analyzed by two-tailed Student’s t test.

Nontransformed NMuMG cells and KRASG12D-expressing cells (3.0 × 10⁴ cells) were seeded with silicone culture inserts (Ibidi). After cells formed a confluent layer, the culture inserts were removed, and cell migration and collision were observed via two CytoWatcher FL microscopes every 10 min for live imaging. Cells were stained with Hoechst 33342.

Migration was assessed using silicone culture inserts (ibidi GmbH, Munich, Germany) in 12-well culture plates. Inserts had two 70 μl wells, both of which were used to plate cells. PTEC, HUVEC or BTEC (1 × 10⁵cells/ml) were plated on 1% gelatin coating in EndoGRO MV-VEGF medium containing 5% FCS. Cells were maintained in incubator until confluence was reached. Cell monolayers were starved 12 hours in DMEM 0% FCS before removing the inserts and thus generating the "wound area". Floating cells were removed by wash in PBS solution, and monolayers were treated with test conditions (in duplicate). EndoGRO MV-VEGF medium 5% FCS was used as positive control, whereas DMEM 0% FCS served as negative control. Cell migration was imaged with a Nikon Eclipse Ti inverted microscope using a Nikon Plan 4X/0, objective and cells were kept on a stage incubator at 37°C and 5% CO2 during the experiment (OKOLab, Italy). Images were acquired at 2 h time intervals using MetaMorph software.
MetaMorph software was used to calculate migration rate (%) by measuring the distance covered by cells between two subsequent time points (4 fields measurements for each image). Measurements were made for each time point and at least 10 fields for each condition were analyzed in each independent experiment. Graphs show mean ± S.E.M of three independent experiments.