MTLn3 cell cotransfected with control siRNA and GFP-cortactin to visualize invadipodia dynamics. The corresponding FAK knockdown experiments are CIL:25705 and CIL:25704. Fluorescence imaging was performed using a 60×/1.40 oil objective on an inverted microscope (1X-70;Olympus) in a 37°C closed system as previously described. Glass-bottomed dishes (35 mm) were coated with 10 µg/ml fibronectin for 1 h at 37°C. Cells were plated in Ham’s F12 containing 5% FBS and 20 mM Hepes, pH 7.2, and were allowed to adhere for 3 h. Fluorescent images were collected using a cooled CCD camera (CoolSNAP FX; Hamamatsu Photonics) and captured into MetaVue every 1 min for 1 h. Time-lapse sequences from live fluorescence imaging were first subjected to high-pass filtration based on the water algorithm (Zamir et al., 1999) to remove diffuse background fluorescence. Video corresponds to Fig. 1 D. and supplemental video 1 in J Cell Biol. 2009 Apr 20;185(2):357-70. Epub 2009 Apr 13. Bar, 10 µm.
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