Nipkow disk confocal scanner unit csux1

HeLa cells (in glass bottom dishes) were cotransfected with Gag-EGFP and mChr-Syx6 or mChr-Syx12 expression plasmids. In some experiments, HeLa cells were cotransfected with Gag-EGFP and mSB-Syx6 expression plasmids and siRNA. For disruption of microtubules, cells were treated with 10 μg/ml. For microtubule-dependent trafficking, EGFP-Syx6 and Gag-EGFP expression constructs were cotransfected with mSB-tubulin expression plasmid. For TNFα expression, Gag-mSB and mSB-Syx6 were coexpressed with TNF-EGFP in HeLa cells. The cells were incubated at 37 °C in a microscope stage top incubation chamber. Live-cell imaging was performed at 20 to 24 h posttransfection with a confocal microscope (IX71, Olympus Optical, Japan) equipped with a microlens-enhanced Nipkow-disk confocal scanner unit (CSUX1, Yokogawa Electric, Japan). Sequential Images (excitation at 488 and 568 nm) were acquired at 1-s intervals for 100 s with an electron-multiplying CCD camera (Luca, Andor Technology). Bleach and contrast corrections of images were performed using ImageJ software, and tracking of punctate fluorescent signals using MTrackJ plugin created by Eric Meijering (http://www.imagescience.org/meijering/software/mtrackj).

Madin-Darby canine kidney cells were seeded in ϕ3.5 cm glass bottom dishes and were co-transfected with HA-EGFP and NA-mSB expression plasmids. MDCK cells stably expressing AcGFP-Rab were similarly cultured in ϕ3.5 cm glass bottom dishes and were transfected with the NA-mSB expression plasmid. Before live-cell imaging, the culture medium was replaced with Dulbecco’s modified Eagle’s medium without phenol red (Life Technologies) supplemented with 10% FBS and the cells were placed in a microscope stage top incubation chamber (Tokai HIT, Japan). Live-cell imaging was performed at 12 hpt using a confocal microscope (IX71, Olympus Optical, Japan) equipped with an oil immersion objective (Plan Apo N, 60×, 1.42NA, Olympus Optical) and a microlens-enhanced Nipkow-disk confocal scanner unit (CSUX1, Yokogawa Electric, Japan). Sequential images with excitation at 488 and 568 nm were acquired at 1-s intervals for 100 s (100 exposures each for GFP and RFP) by an electron multiplying CCD camera (Luca, Andor Technology, United Kingdom). Bleach and contrast corrections of acquired images were performed using ImageJ software, and tracking of punctate fluorescent signals using MTrackJ plugin created by Eric Meijering¹.