Phalloidin conjugated with alexa fluor 488

Cells were cultured in Petri dishes until the desired density of cells was obtained, i.e., single cells or cell monolayers. Cells were washed with PBS buffer and fixed by adding a 3.7% paraformaldehyde solution in PBS for 20 minutes. Next, they were washed with PBS three times, and the cell membrane was permeabilized using 0.2% Triton X-100 (Sigma) in PBS at 4° C for 5 minutes. Afterwards, the cells, rinsed with PBS, were labelled with fluorescent dyes in an environment with a minimum light level. Phalloidin conjugated with Alexa Fluor 488 (Invitrogen) was dissolved in PBS buffer (1:200) and used to stain the actin filaments for 30 minutes, while the cell nuclei were stained with Hoechst 33342 (Invitrogen) dissolved in PBS buffer (1:5) for 15 minutes. Afterwards, the samples were washed with PBS buffer. The fluorescent images were recorded using an Olympus IX83 inverted microscope (Olympus, Japan) equipped with a 100 W mercury lamp and a set of filters used to record the emission at 515-545 nm (Alexa Fluor 488) and 461 nm (Hoechst 33342). The images were acquired using the Orca Spark digital camera (Hamamatsu, Japan), providing 2.3 megapixels (1920 pixels per 1200 pixels) images that were recorded using CellSens Dimension software (Olympus).