Floid cell fluorescence imaging station

For cell morphologies of HepG2 and CCL-13 before and after incubation with chitosan nanoparticles, the cell live images were observed with a microscope equipped with fluorescence light source (FLoid cell fluorescence imaging Station, Invitrogen), and the cell micrographs were taken with a CCD camera.

The SH-SY5Y cells were grown on coverslips in 12-well culture plates. After 24 hours' incubation, the cells were fixed (60% methanol and 40% acetone) at −20°C for 30 min and then permeabilized (0.5% Triton X-100) at room temperature for 5 min. After rinsing with PBS, the cells were blocked (6% bovine serum albumin) and then incubated with primary and secondary antibodies. The nuclei and cytoskeleton of the cells were stained with DAPI (Sigma-Aldrich, USA), vimentin (Vimentin DyLight 488 Antibody, Epitomics, USA), monoclonal mouse anti-HSP60 (Stressgen, USA), and polyclonal rabbit anti-RanBP2 (Abcam, USA), respectively. After rinsing with PBS, the cells were mounted with ProLong® Gold Antifade Reagent (Invitrogen). The images were acquired by a microscope equipped with fluorescence light source (FLoid Cell Fluorescence Imaging Station, Invitrogen).

For cell morphology of adhered fibroblasts on the aforementioned electrodes after incubation, the electrodes were washed and fixed with 4% formaldehyde at 4°C. The nuclei and cytoskeleton of the cells were stained with 4′-6-diamidino-2-phenylindole (DAPI, 32670, Sigma-Aldrich, USA) and vimentin (Vimentin DyLight 488 Antibody, Epitomics, USA), respectively. In addition to staining with DAPI and vimentin, the anti-CD44 antibody (1998-1, Epitomics, Inc) was incubated and followed by staining with Alexa Fluor 568 goat anti-rabbit IgG (A-11011, Invitrogen). The samples were blocked with 2% bovine serum albumin (BSA, A1933, Sigma-Aldrich, USA) at room temperature for 30 min. The cell images were observed by a microscope equipped with fluorescence light source (FLoid Cell Fluorescence Imaging Station, Invitrogen), and the cell micrographs were taken with a CCD camera.