Inverted evosmicroscope

Cell migration was evaluated using a modified wound scratch assay. The cells were plated (5 × 10⁴ for HT29; 3 × 10⁴ for HCT116; 4 × 10⁴ for HCT116 p53−/− cells) in a cell culture dish (Ibidi, Martinsried, Germany) and, when cell confluence was approximately 90%, the chambers were removed and the cells were treated with metformin. The images (magnification 10x) were acquired immediately after removing the chambers, and at the complete closure of the gap in the untreated cells using an inverted EVOSmicroscope (Thermofisher Scientific, Inc., Waltham, MA, USA).
Cell invasion was evaluated by plating 3 × 10⁴ cells per well in a 24-well BDBioCoat growth factor-reduced MATRIGEL invasion chamber (BD Biosciences, Franklin Lakes, NJ, USA), and treating them with metformin. Inserts were placed in a well containing 750 µl of normal growth medium with 10% FBS. After 72 hours (HCT116 and HCT116 p53−/− cells) or 96 hours (HT29 cells), the cells in the upper compartment were removed, and the invading cells attached to the bottom side were fixed with cold methanol and stained with Crystal Violet (Sigma-Aldrich). Three independent experiments were carried out.

Cell migration was assayed using a Boyden‐modified chamber (transwell inserts with 8‐µm pore size were precoated with Matrigel, 0.75 mg·mL⁻¹; Becton Dickinson, Franklin Lakes, NJ, USA) as described previously [30 , 31 (link)]. Briefly, cells were detached with trypsin/EDTA and washed twice in serum‐free growth medium by centrifugation (300 g, 3 min). Cells (6 × 10⁴) in 200 μL of serum‐free growth medium were transferred to the upper chamber whereas inhibitor‐ or vehicle‐containing complete medium (500 µL) was transferred to the lower chamber. Multiple wells and multiple experiments were performed for reproducibility. Cells were fixed and stained using Diff‐Quick II staining kit (Dade Behring, Marburg, Germany) per the manufacturer's instruction after a 24‐h incubation period at 37 °C. Images were acquired under an inverted EVOS microscope (Thermo Scientific).