Scratcher

The effects of compounds 1–5 on HUVEC migration were determined using the JuLI™ Stage system (NanoEntek, Seoul, Korea): a real-time cell history recorder allowing for live cell imaging and analysis [74 (link)]. The procedure for the migration experiments has been published elsewhere in more detail [74 (link)]. Concisely, HUVECs were seeded at a density of 10,000 cells per well on 96-well plates and incubated at standard conditions for 24 h. Following this, a wound was made with a scratcher (NanoEntek, Seoul, Korea). The cells were then rinsed with 100 μL of fresh EGM2 medium, and treated with biguanides 1–5 at the concentrations of 0.3 and 1.0 mM. The cells were incubated and monitored continuously every 10 min for 36 h at 37 °C, 5% CO₂. Analysis of obtained images of cell migration, and measurements of the width of the scratch area were performed using dedicated software (NanoEntek, Seoul, Korea). The results were presented as mean ± SD, n = 4–8. The CV for the method was estimated as 14.2–24.4%, depending on the time point (n = 8).

HUVEC cells were cultured on 96-well plates and incubated until reaching 70% confluence. After culturing for 24 h, the confluent cells were wounded by scratching with a manufacturer provided scratcher (NanoEntek, Seoul, Korea), and the wells were rinsed with 100 μL of fresh medium. Afterwards, medium was replaced with the same volume of fresh medium (control) or medium including compounds at various concentrations (10 + 90 μL, final concentration of compounds 0.1 and 0.5 μmol/mL). The plates were incubated up to 36 h at 37 °C (5% CO₂). In this period, migration of cells was monitored using the JuLI™ Stage system which is a Real-Time CHR (Cell History Recorder, NanoEntek, Seoul, Korea) designed for live cell imaging and analysis. The images of cells migration were acquired continuously every 10 min (high-sensitivity monochrome CCD (Sony sensor 2/3”, Tokyo, Japan)). The images were analyzed by dedicated software (NanoEntek, Seoul, Korea), and the width of the scratch area was measured. The results are presented as mean ± SD, n = 4–8. The coefficients of variation for the applied method were determined (CV = 13.6–16.0%, depending on the time point, n = 8)