C apochromat 40 1.2 w

Cell spreading was assessed on all surfaces after 20 min and 24 h, respectively. In total, 10⁶ cells were suspended in 250 µL diluent C and their cell membranes were stained with PKH-26, a lipophilic membrane dye (PKH-26 general cell linker kit, Sigma-Aldrich, Steinheim, Germany) for 5 min at 37 °C using a dilution of 2 µL PKH-26 + 248 µL diluent C. After stopping the staining reaction using FCS, cells were washed with Dulbecco’s phosphate buffered saline (PBS), resuspended in cell culture medium and 3 × 10⁴ cells were seeded per specimen. After 20 min or 24 h, cells were rinsed twice with Dulbecco’s phosphate buffered saline (PBS) (Sigma-Aldrich), fixed with 4% paraformaldehyde for 10 min at room temperature (RT), rinsed with PBS and embedded with mounting medium (Fluoroshield with DAPI, Sigma-Aldrich) and a cover slip. Cells were examined with a water immersion objective (C Apochromat 40×, 1.2 W, Carl Zeiss, Oberkochen, Germany) at a wavelength of 546 nm using a confocal laser scanning microscope (LSM780, Carl Zeiss, Oberkochen, Germany; ZEN 2011 software black version, Carl Zeiss, Oberkochen, Germany). The mean spreading area in µm² of 40 cells per specimen was then calculated using image processing software (ImageJ, v2.0.0, National Institutes of Health, Bethesda, Maryland, USA).