Nucblue live cell reagent

Chemotactic capacity of RPE-1 cells was monitored using 6-well Transwell permeable supports with 8 μm pores (Corning, Cat. 3428). Cells were detached by trypsinization, washed 2× in PBS, suspended at the concentration of 2 × 10⁵/ml in DMEM (Lonza, Cat.BE12-614F) plus 0.1% FBS (Euroclone, Cat. ECS0181L) and seeded in the above inserts (final cell number: 3 × 10⁵). Chemotactic migration was induced by adding DMEM (Lonza, Cat.BE12-614F) + 10% FBS (Euroclone, Cat. ECS0181L) + 50 ng/ml human recombinant epithelial growth factor (Vincil-Biochem, Cat. BPS-90201-3) in the bottom reservoir (2 ml of total volume). After 6 h of incubation at 37 °C, migrating cells were stained with NucBlue® Live Cell reagent (ThermoFisher, Cat. R37605). The cells on the upper side of the insert were scraped using a cotton swab. The cells present on the bottom side were imaged using widefield BX63 Olympus equipped with 4x objective. The total area of the well was scanned and the average number of cells per area was calculated.

3 × 10⁵ hTERT_RPE1 cells were seeded in six-well plates over-night before live-microscopy analysis. Then, cells were stained with NucBlue Live Cell Reagent (Thermo Fischer) and imaged every 10 min for 18 h using a humidity- and temperature-controlled inverted wide-field microscope within an environmental chamber (ScanR, Olympus). Nuclear tracking and cell motility analysis were performed as described here^{20 (link)}. For this purpose, specific software in C++ with the OpenCV [http://opencv.willowgarage.com/wiki/] and the GSL [http://www.gnu.org/software/gsl/] libraries were developed. The migration analysis was performed by the C++ software coupled with R [www.R-project.org].