Eclipse ti inverted light microscope

A Matrigel-based Transwell system (8-mm pore size polycarbonate membrane; Corning, Inc.) was used to examine the invasiveness of transfected cells. The Transwell inserts were covered with a homogenous layer of BD Matrigel™ Basement Membrane Matrix (BD Biosciences) and incubated for 4 h at 37˚C in 24-well plates containing RPMI-1640 medium per 10-20 µl/well. Then, 0.3-1.0x10⁵ cells/well in 250.0 µl serum-free medium were plated into the upper chamber and allowed to invade for 24-72 h at 37˚C. A total of 650.0 µl of RPMI 1640 medium containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) was plated in the lower chamber. The residual medium on the top insert was removed after the incubation time and non-invading cells on the upper surface were gently scraped with a brush. The migrating cells to the lower chambers were fixed in a fixing solution (4% formaldehyde in PBS with 0.1% Tween-20) at room temperature for 20 min after being washed twice with PBS. The cells were then stained for 10 min at 37˚C with 4',6-diamidino-2-phenylindole solution (Sigma-Aldrich; Merck KGaA) and photographed using a microscope Nikon ECLIPSE Ti inverted light microscope (Nikon Corporation) at 100x or 200x magnification. Subsequently, the number of invasive cells was by three fields per chamber using Image J software 1.52d (National Institutes of Health).

The two-well silicone insert (Ibidi GmbH) was utilized to perform the gap closure experiment. Adhesive inserts with sticky floors were pasted onto six-well plates before performing the experiments. After transfection, the MCF-7, MDA-MB-231 cells (5.0-7.0x10⁵ cells per each side of the insert) were reseeded into a two-well silicone insert with 70.0 µl of cell suspension and cultured for 24 h at 37˚C to create a 500 µm gap in a confluent cell monolayer. The inserts were then gently removed with sterile tweezers and immediately taken under a microscope (Nikon ECLIPSE Ti inverted microscope; Nikon Corporation) to collect sample images at timepoint zero. The plates were rinsed several times in PBS and incubated with fresh RPMI 1640 medium containing 10% serum (Gibco; Thermo Fisher Scientific, Inc.) for 17 or 30 h at 37˚C. At different time points, the plates were taken under a Nikon ECLIPSE Ti inverted light microscope (magnification, 40x; Nikon Corporation). In the images taken using the microscope, the area of the gap was marked and assessed using ImageJ 1.52d (National Institutes of Health). Data were normalized to the mean value at time zero.