Ff01 542 27 25 emission filter

HeLa/Fucci2 cells were grown on 35 mm glass-bottom dishes in phenol red-free DMEM containing 10% FBS and transfected with either pME18Neo/Flag-Vpr-IRES-ECFP or the control pME18Neo/Flag-IRES-ECFP. The cells underwent long-term, time-lapse imaging using a computer-assisted fluorescence microscope (Olympus, LCV110) equipped with an objective lens (Olympus, UAPO 40X/340 N.A. = 0.90), a halogen lamp, a red LED (620 nm), a CCD camera (Olympus, DP30), and interference filters. For fluorescence imaging of FRET, we used the halogen lamp with a BP425-445HQ excitation filter (Olympus), a DM450 dichroic mirror (Olympus), and an FF01-542/27-25 emission filter (Semrock) for observing SCAT3.1. To quantify the results, the images of ECFP and Venus fluorescence were processed with MetaMorph 7.7.4 software (Universal Imaging), and the Venus/ECFP emission ratio was calculated.

CFP and YFP images of HeLa cells and A549 cells were obtained by using an inverted microscope (IX81-ZDC; Olympus, Tokyo, Japan) equipped with a cooled CCD camera (Cool SNAP-K4; Roper Scientific), an illumination system (Spectra-X light engine; Lumencore, OR), an IX2-ZDC2 laser-based autofocusing system (Olympus), a MAC5000 controller for filter wheels and XY stage (Ludl Electronic Products, Hawthorne, NY), an incubator chamber system (Tokai Hit, Shizuoka, Japan) and a GM-4000 CO2 supplier (Tokai-Hit, Fujinomiya, Japan). The following filters were used for the dual emission imaging studies: FF01–438/24–25 (Semrock, Rochester, NY, USA), and FF01–475/28–25 (Semrock) excitation filter for CFP and YFP/GFP, a U-MREF glass reflector (Olympus) as a dichroic mirror, an FF01–483/32–25 emission filter (Semrock) for CFP and an FF01–542/27–25 emission filter (Semrock) for YFP. The microscope was controlled by MetaMorph software (Universal Imaging, West Chester, PA). The average fluorescence intensities of CFP and YFP in each cell were measured by manually delineating a region of interest at the cytoplasm with MetaMorph software.

HeLa/Fucci2 cells were grown on 35 mm glass-bottom dishes and transfected with the indicated vectors or infected with adenoviral vector expressing Vpr and ZsGreen1 virus. At the indicated times after transfection or infection, cells were subjected to long-term time-lapse imaging using a computer-assisted fluorescence microscope (Olympus, LCV110) equipped with an objective lens (Olympus, UAPO 40X/340 N.A. = 0.90), a halogen lamp, a red LED (620 nm), a CCD camera (Olympus, DP30), and interference filters. For fluorescence imaging of Fucci2 with ECFP or ZsGreen1, the halogen lamp was used with an FF02-510/10-25 excitation filter (Semrock, Inc., Rochester, NY), an FF520-Di02-25x36 dichroic mirror (Semrock), and an FF01-542/27-25 emission filter (Semrock) for Venus; an FF01-562/40-25 excitation filter (Semrock), an FF593-Di03-25x36 dichroic mirror (Semrock), and an FF01-641/75-25 emission filter (Semrock) for mCherry; and a CFP2432B filter cube (Semrock) for ECFP; an FF02-472/30-25 excitation filter (Semrock), and an FF495-Di03-25x36 dichroic mirror (Semrock), and an FF01-520/35-25 emission filter (Semrock) for ZsGreen1. Image acquisition and analysis were performed using MetaMorph 7.7.4 software (Universal Imaging, Media, PA). Movies were assembled using QuickTime software.