Fluoromount

Similar to our previous report^{35 (link)}, to examine the cell morphology under confocal microscopy after 24 h of incubation, cells on each surface were fixed in 4% paraformaldehyde for 20 min. A 1 μL stock solution of phalloidin-488 (AAT Bioquest) was diluted in 100 μL of PBS containing 1% bovine serum as the main working solution for staining. The surfaces were also washed with PBS for three times before staining the nuclei with 1 μL of Hoechst 33342 (AAT Bioquest) in 1 mL of PBS buffer for 10 min. Upon completion of staining and washing, a drop of Fluoromount (NOVUS) was carefully aliquoted onto the surface and subsequently covered with a coverslip and sealed with nail varnish. The stained cells with actin filament were observed under confocal microscopy (LEICA SP8) at 40x magnification to examine the cell morphology.

To evaluate cell number and morphology, cells on each surface were fixed in 4% paraformaldehyde for 20 min. A 1 μL stock solution of phalloidin-594 (AAT Bioquest, Sunnyvale, CA, USA) was diluted with 100 μL of PBS containing 1% bovine serum as the working solution to stain the cells’ actin filaments. To ensure no excess phalloidin remained, the surfaces were also washed with PBS three times, followed by staining of the nuclei with 1 μL of Hoechst 33342 (AAT Bioquest) in 1 mL of PBS buffer for 10 min. Upon completion of staining, a drop of Fluoromount (NOVUS) was added to the surface, which was then covered with a coverslip and subsequently sealed with nail varnish. The cells with stained actin filaments were observed by confocal microscopy (LEICA SP8) at 40× magnification to examine the cell morphology. To perform the cell count analysis, a total of n = 5 surfaces were studied for each condition selected, and five random spots were chosen on each of the surfaces. Fluorescence microscopy images were taken for each of the random spots, and the number of cells was counted. All data were tabulated, and their standard derivations calculated accordingly, in an Excel spreadsheet.