Live cell imaging

Live cell imaging was performed for invadopodia lifetime, cortactin oscillations, and calcium spike analysis. In brief, Cerulean-cortactin-MTLn3 cells were cultured on MakTek dishes coated with Alexa 488 or 546 gelatin and incubated in culture conditions for 16 h. For lifetime analysis and cortactin oscillations, the media were changed to L-15 with 5% FBS, 1:100 Oxyfluor (Oxyrase, Mansfield, OH), and 10 mM sodium lactate (Sigma-Aldrich) as reactive oxygen scavengers. Dishes were placed in a 37°C live-cell imaging chamber, and time-lapse imaging was performed on a widefield Olympus IX81 microscope (Olympus) equipped with an LED lamp, Hamamatsu Orca 16-bit charge-coupled device (Hamamatsu, Hamamatsu, Japan), automated z-drift compensation IX3-ZDC (Olympus), automated Prior stage (Prior Scientific, Rockland, MA), and environmental chamber. An Olympus 60X 1.4 NA Oil M Plan Apochromat objective was used.