Fluorescence recovery after photobleaching (FRAP) experiments

Cells were incubated for approximately 30 min with lipophilic dye BODIPY FL C12 (Molecular Probes, Eugene, OR). Fluorescence microscopy was carried out using a Nikon X1 spin flask confocal microscope (Nikon), and images were acquired using a digital black and white camera (Hamamatsu, Bridgewater, NJ) and NIS-Elements software (Nikon, Melville, NY). Cells were cultured in a chambered coverslip and placed on a temperature-controlled stage under the microscope objective lens. A 60x oil immersion lens was used. The confocal spot was scanned for about 0.5s in the middle of the cell to create a circular bleach zone. The confocal spot was then scanned to record a sequence of images of the cell at 0.5-s intervals. Images were analyzed using ImageJ with the simFRAP plugin [25].