Hoechst 33258 dna dye

To study the cell adhesion on CS/(HA/COL)5 coating, CS/(HA/COL)5-AdGFP coating, and CS/(HA/COL)5-AdLAMA3 coating and the smooth titanium surface, HaCaT cells were seeded on the 8 mm² × 8 mm² plates at a density of 2 × 10⁴/well in 48-well plates. After 1, 3, and 6 h of incubation, the samples were rinsed with 0.01 M of PBS 3 times to remove the unattached cells, and then fixed by 4% paraformaldehyde at 4°C for 20 min. After being rinsed with PBS, the samples were incubated with 0.5% bovine serum albumin for 30 min to prevent non-specific binding. The cell actin microfilaments were then stained using rhodamine labeled phalloidin (Cytoskeleton Inc., United States) at a 1:40 dilution. After being rinsed three times with PBS, nuclei were stained with Hoechst 33258 DNA dye (Beyotime, China) for 5 min. Finally, the samples were observed and photographed with a fluorescence microscope (Eclipse-80i; Nikon, Tokyo, Japan). The cell number was measured in 10 randomly selected areas on each sample using Image-Pro version 6.0 software (Media Cybernetics Corp., United States), three samples in each group.

Plasmids, specifically pReceiver-M61C-LAMC2, were constructed by GeneCopoeia (USA) and amplified at the School of Basic Medical Science, Zhejiang University. Chitosan (CS, molecular weight, 100,000) was purchased from Qingdao Yunzhou Biochemistry (China). Fluorescein-isothiocyanate-labeled phalloidin, Triton X-100, CelLytic buffer, and hyaluronic acid (HA) were purchased from Sigma Chemical Co. (MO, USA). A Label IT^® TM-Rhodamine labeling kit was purchased from Mirus Bio, LLC. (USA). Alamarblue and Lipofectamine LTX Plus reagent were purchased from Invitrogen (USA). Bovine serum albumin was purchased from Shanghai Sangon Biological Engineering Technology and Services (China). Hoechst 33258 DNA dye was purchased from Beyotime (China). Anti-laminin γ2 mouse monoclonal antibody, anti-integrin β4 mouse monoclonal antibody and anti-plectin rabbit monoclonal antibody were purchased from Abcam (USA). Goat anti-mouse IgG conjugated with Alexa 488 and goat anti-mouse IgG conjugated with Alexa 594 were purchased from Invitrogen (USA). Goat anti-rabbit IgG conjugated with rhodamine was purchased from Jackson ImmunoResearch Laboratories (USA). A human laminin-5 ELISA kit was purchased from R&D Technologies. Flat pure titanium plates 10 × 10 × 1 mm in size were purchased from Zhejiang Guangci Medical Appliance Company (China).