Visualizing F-Actin Polymerization in Cells

To visualize the polymerized F-actin, cells (~2x10⁴/sample) were seeded on glass cover-slips and cultured in growth medium in the absence (control) or presence of 300 µM AdoMet. After 24 and 48 h, the slides were washed with PBS, fixed with 2.5% formaldehyde, permeabilized with 0.1% Triton X-100 for 10 min at 4˚C, and incubated with 0.1 µg/ml rhodamine-conjugated phalloidin (Sigma-Aldrich; Merck KGaA) for 40 min at 23˚C (25 (link)). Nuclear staining was performed with the 10 mM 4-6-diamidino-2-phenylindole (DAPI) dye (#228549, Abcam) for 5 min at 23˚C. Finally, coverslips were mounted using 20% (w/v) Mowiol, and visualized with an Axiovert 200 M fluorescence inverted microscope connected to a video camera (Carl Zeiss AG).