2.7. Cell staining
Live/Dead staining assay was conducted to determine viability of cultured cells. Calcein AM (Invitrogen, USA, C34852) and ethidium homodimer-1 (Invitrogen, USA, E1169) were diluted to 4 μM and 2 μl of each solution was added into 1 ml of phosphate-buffered saline (PBS; Sigma, USA, P3813). Cells were incubated with Live/Dead solution for 90 min. F-actin and nucleus were stained to assess formation of cell monolayer. First, cells were fixed by 4% paraformaldehyde (Sigma, USA, 8187081000) for 10 min. Next, fixed tissue was treated with 0.5% Triton-X 100 (JUNSEI, Japan, 49415-1601) for 3 min. Then, the tissue was stained by FITC-phalloidin (Sigma, USA, P5282) and 0.1 μg/ml of DAPI (Sigma, USA, 10236276001) for 30 and 1 min respectively. The Images were taken by a fluorescence microscope (Olympus, Japan, CKX41) or a confocal microscope (Carl Zeiss, Germany, LSM 880).