Wound Healing Assay with Dynamin Inhibitors

H1299 cells were cultured to confluence on glass-bottom dishes (35 mm diameter; AGC Techno Glass Co. Ltd., Tokyo, Japan) in DMEM supplemented with 0.2% FBS for 8 h. Thereafter, the cell layer was wounded with a plastic pipette tip as previously described (25 (link)). The cells were washed with DMEM supplemented with 0.2% FBS and incubated for 8 h in the presence of Dynasore, Dynole 34-2 or MitMAB at the indicated concentrations. For the negative control, cells were incubated with 1% dimethyl sulfoxide (DMSO). The cells were visualized by Giemsa staining, followed by the acquisition of phase contrast images from ≥20 randomly selected areas per dish. Areas filled with migrating cells were analyzed with ImageJ software (National Institutes of Health, Bethesda, MD, USA).