FRET Analysis of Cellular Dynamics

FRET analysis was performed according to the previously published method with minor modification^{26 (link)}. Briefly, FRET signals were imaged with a DeltaVision microscope system (GE Healthcare) built on an Olympus IX-71 inverted microscope base equipped with a Photometric Coolsnap HQ2 CCD camera and a 60×/NA1.516 PlanApo oil immersion lens (Olympus). For live-cell imaging FRET sensors, cells were seeded on gelatin-coated CELLview Cell Culture Dishes (Greiner Bio-One) and maintained in an incubator at 37 °C with 5% CO₂. For imaging, cells were observed with a Blue excitation filter (400-454 nm), two emission filters (blue-green, 463–487 nm for ECFP; yellow-green, 537–559 nm for Ypet), and a C-Y-m polychronic mirror. The FRET emission ratio (FRET/CFP) was calculated with SoftWoRx (Applied Precision Inc) by dividing the excitation at 436 nm and emission at 560 nm (FRET) by the excitation at 436 nm and emission at 470 nm (CFP). For statistical analyses, the obtained images were analyzed with ImageJ and MetaMorph software. The ΔFRET/CFP ratios were calculated by subtracting the FRET/CFP ratio at time 0 from the FRET/CFP ratio at the indicated times.