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 .
Electron multiplying ccd camera
Manufactured by Oxford Instruments
Sourced in United Kingdom
The Electron Multiplying CCD Camera is a specialized imaging device that uses an electron-multiplying charge-coupled device (EMCCD) sensor. It offers high-sensitivity, low-noise performance, and is designed for applications that require the detection of low-light signals.
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Lab products found in correlation
3 protocols using electron multiplying ccd camera
1
Live-Cell Imaging of Rab-Mediated Trafficking
2
Multimodal Imaging of Cellular Dynamics
3
Syp1-downregulated PC12 Cell Lines
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Stable, clonal lines of Syp1-downregulated PC12 cells were generated using different types of shRNA coding sequences for mouse Syp1 (OriGene) according to the manufacturer's protocol (for further details, see Supplemental Experimental Procedures). For immunofluorescence analysis, cells were fixed with 4% (w/v) paraformaldehyde in PBS for 20 min. After washing, cells were quenched with 0.1 M glycine for 20 min and permeabilized for 5 min with 0.2% (v/v) Triton X-100 in PBS. Cells were stained with the respective antibodies diluted in PBS containing 1% (w/v) BSA at RT (2 hr for primary antibodies and 1 hr for secondary antibodies). Coverslips were mounted in PBS. Images were taken using TIRF microscopy performed on a Nikon Ti Eclipse using fiber-coupled lasers of different wavelengths: 491 nm (100 mW Calypso, Cobolt) and 561 nm (100 mW Jive, Cobolt). Fluorescence emission was detected using a 3100 oil objective (Nikon Apo TIRF, NA 1.49) together with an electron-multiplying CCD camera (Andor Technology).
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