Quantitative Analysis of F-Actin Dynamics

The cells were seeded onto the glass-bottom of fibronectin (FC010, Millipore, Temecula, CA, USA) coated culture dishes (P35GC-014-C; MatTek, Ashland, MD, USA). Following the 36 h attachment, the cells were fixed with 3.7% paraformaldehyde, and then, permeabilized with 0.1% Triton X-100 in phosphate buffered saline (PBS) at room temperature for 10 min. The cells were washed with PBS and blocked in a 1% bovine serum albumin (BSA)/PBS solution for another 1 h. The F-actin protein expression was revealed by its incubation with Texas Red X-Phalloidin (Invitrogen) and the immunofluorescence was recorded using a confocal microscope (LSM510 Meta, Zeiss, Oberkochen, Germany), as described previously [20 (link)]. The F-actin fluorescence intensity was assessed using Zen blue edition software (version 3.2, Zeiss). The intensity profiles were measured along the line from the peripheral to the central of the cells.