MTLn3 cell cotransfected with FAK siRNA (target A) and two tandem Src SH2 phosphotyrosine-binding domains (YFP-dSH2), which specifically detects tyrosine phosphorylation at focal adhesions. CIL:25703 is the corresponding control experiment. 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. Movie corresponds to video 5 from J Cell Biol. 2009 Apr 20;185(2):357-70. Epub 2009 Apr 13.
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