Fluorescein isothiocyanate conjugated phalloidin

The cytocompatibility of the ternary composite mat was assessed via MTT assay. In brief, coverslips and scaffolds were fixed in 24-well plates with stainless-steel rings and sterilized by exposure to 75% alcohol solution for 2 hrs. After that, all of the samples were washed three times with PBS solution and soaked in DMEM overnight before cell seeding. Then, mouse fibroblast cells (L929) were seeded at a density of 1.5 × 10⁴ cells per well for 2, 4, 8, and 12 hrs for cell adhesion assay and 1, 3, 5, and 7 d for cell proliferation assay. Coverslips without scaffolds were used as controls.
MTT assay and SEM observation were employed to separately evaluate the viability and morphology of the adhered and proliferated L929 cells cultured onto different scaffolds (n=3 for each group) according to the manufacturer’s protocol of our previous research.26 (link) On a separate set of sheets, the attached cells were fixed with 4% paraformaldehyde and then 4′,6′-diamidino-2-phenylindole hydrochloride (DAPI, Invitrogen, USA) and fluorescein isothiocyanate-conjugated phalloidin (Invitrogen, USA) were used to stain the nucleus and cytoskeletons of cells. Specimens were observed under a TS100 fluorescence microscope (Nikon, Japan).

To assess the morphological variation of BASCs induced by the substrates, we performed fluorescence staining of F-actin. In brief, after 1 and 3 days of incubation at 37°C and 5% CO₂, the engineered constructs of cells and scaffolds were fixed using 4% paraformaldehyde for 30 minutes at room temperature, and then stained with fluorescein isothiocyanate–conjugated phalloidin (1:500; Invitrogen) for F-actin and 4,6-diamidino-2-phenylindole (1:1,000; Molecular Probes, MA, USA) for the cell nucleus. The results were observed by confocal microscopy. To determine the potential effects of CNTs on cell adhesion of BASCs, ten pictures per section under 20 × fields of BASCs on CNT-Col or Col substrates were randomly acquired for analysis using ImageJ software (National Institute of Health). Additionally, to quantify the cell shape change due to the incorporation of CNTs, the cell area and the long/short axis of BASCs at days 1 and 3 were analyzed with ImageJ software. An average of 300 cells was counted for each sample group.