Ultraview lci

Manufactured by PerkinElmer

The Ultraview LCI is a lab equipment product offered by PerkinElmer. It is designed to perform live-cell imaging. The core function of the Ultraview LCI is to capture and analyze images of living cells over time.

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Lab products found in correlation

Orca er, by Hamamatsu Photonics (3 mentions) Ab91506, by Abcam (1 mentions) Vectashield dapi mounting media, by Vector Laboratories (1 mentions) Alexa fluor secondary antibody, by Thermo Fisher Scientific (1 mentions) Kaleidagraph 4, by Synergy Software (1 mentions) Metamorph 7, by Molecular Devices (1 mentions) Er tracker green, by Thermo Fisher Scientific (1 mentions) Ultraview imaging suite software, by PerkinElmer (1 mentions) Oregon green 488 bapta 5n, by Thermo Fisher Scientific (1 mentions) E600fn, by Nikon (1 mentions) Lambda 10 2 filter wheel, by Sutter Instruments (1 mentions) Nis elements ar software, by Nikon (1 mentions) Deltavision, by Cytiva (1 mentions)

6 protocols using ultraview lci

Immunofluorescent Myosin Heavy Chain Assay

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Coverslips were rinsed with PBS and fixed in 4% paraformaldehyde for 10 min. They were then washed in PBS, incubated in PBS supplemented with 1% bovine serum albumin and 0.1% triton X-100 (permeabilization solution) for 20 min, before being blocked for 30 min in the permeabilization solution + 5% donkey serum (blocking solution). The fixed cells were incubated in blocking solution overnight at 4°C with either a primary antibody against MyHC all classes (A4.1025, IgG, Developmental Studies Hybridoma Bank), diluted 1:10, a MyHC slow antibody (Sigma M8421), diluted 1:500, or a MyHC fast antibody (abcam ab91506), diluted 1:1000. Coverslips were washed with PBS and incubated with Alexafluor secondary antibodies (Invitrogen) diluted (1:400) in PBS for 2 hours at ambient temperature in the dark. After rinsing in PBS, the coverslips were mounted on glass slides using VectaShield + DAPI mounting media (Vector Laboratories H1200) and viewed on a confocal microscope (UltraVIEW^™ LCI, PerkinElmer).

McAleer C.W., Rumsey J.W., Stancescu M, & Hickman J.J. (2015). Functional Myotube Formation from Adult Rat Satellite Cells in a Defined Serum-free System. Biotechnology progress, 31(4), 997-1003.

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Quantitative Analysis of Kinetochore Proteins

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Yeast cells were imaged with a spinning-disk confocal microscope (Ultraview LCI; Perkin-Elmer) equipped with 100× N.A. 1.40 Plan-Apochromat oil immersion objective, and an electron-multiplying charge-coupled device camera (ORCA-ER; Hamamatsu Photonics) was used to capture the real-time images. All imaging was carried out in a 26 °C incubator. Detailed imaging conditions are provided in Table S4.
The imaging data were processed by MetaMorph 7.7 (Molecular Devices). The intensity of Mis12 and Cnp1 at kinetochores was measured using the Region Measurement function in Metamorph 7.7. In brief, a maximum intensity projection of the images was performed, and the intensities of Cnp1 and Mis12 at the moment of chromosome segregation were measured. The spindle lengths were measured using Metamorph 7.7 and the MTrackJ plug-in in ImageJ (National Institutes of Health). Detailed information regarding data analysis was previously described (58 (link)).
Box plots and graphs were generated with KaleidaGraph 4.5 (Synergy Software). Student’s t test was performed using Microsoft Excel for comparison between means for data analysis.

Zhou X., Zheng F., Wang C., Wu M., Zhang X., Wang Q., Yao X., Fu C., Zhang X, & Zang J. (2017). Phosphorylation of CENP-C by Aurora B facilitates kinetochore attachment error correction in mitosis. Proceedings of the National Academy of Sciences of the United States of America, 114(50), E10667-E10676.

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Visualizing Intracellular ER in Isolated Rods

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The ER of isolated rods was labeled by incubation with 1 μM ER-tracker green (Invitrogen) for 30 min at 20°C. Terminals of rods were photobleached for 5 s by illumination with a small spot (8 μm diameter) from a 30-mW 488-nm laser. ER-tracker green was illuminated by epifluorescence and images were acquired every 3 s. To show that ER-tracker dye labeled intracellular structures, we also visualized rods using a spinning disk confocal microscope (PerkinElmer Ultraview LCI).

Chen M., Križaj D, & Thoreson W.B. (2014). Intracellular calcium stores drive slow non-ribbon vesicle release from rod photoreceptors. Frontiers in Cellular Neuroscience, 8, 20.

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Ca2+ Imaging in Retinal Photoreceptors

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Ca²⁺ imaging was performed during whole cell recordings in retinal slices by filling photoreceptors with the Ca²⁺ sensitive dye Oregon Green 488 BAPTA-5N (OGB-5N, k_d = 20 μmol/L; Invitrogen) included in the patch pipette solution (500 μmol/L). OGB-5N at this concentration contributes a Ca²⁺ binding ratio (κ’) of ˜20. When included with a pipette solution containing 0.05 mmol/L EGTA, this produces a total Ca²⁺κ’ of ˜50, which is within the range of measurements we previously reported for both rods and cones (Van Hook and Thoreson 2014 (link)). Images were acquired on a Nikon E600FN microscope equipped with a spinning disk laser confocal scan head (PerkinElmer Ultraview LCI) and a cooled CCD camera (Orca ER) with UltraView Imaging Suite software (PerkinElmer). OGB-5N was excited with a 488-nm laser and emitted light passed through a 525-nm long-pass filter. The frame was cropped and binned 2 × 2 to the synaptic terminals to give quick acquisition speeds of 35 msec exposures at 35 msec intervals. Ca²⁺-dependent fluorescent signals were analyzed as ΔF/F_o (measured from a 1–2 sec prestimulus baseline) in response to depolarizing steps in voltage clamp (–79 to –19 mV) of varying duration (20–500 msec).

Van Hook M.J, & Thoreson W.B. (2015). Weak endogenous Ca2+ buffering supports sustained synaptic transmission by distinct mechanisms in rod and cone photoreceptors in salamander retina. Physiological Reports, 3(9), e12567.

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Imaging Release from Retinal Slices

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For studies of release from retinal slices, pieces of isolated retina were loaded by incubation in darkness with SR101 (83 μM, 30 min), 3-kDa Texas Red (133 μM, 45 min), 10-kDa Texas Red (100 μM, 60 min), or 70-kDa Texas Red (36 μM, 60 min). After incubation, retina pieces were washed in Ca²⁺-free, high-Mg²⁺ saline and slices were prepared as described above. The recording chamber was placed on an upright fixed-stage microscope (Nikon E600 FN) equipped with a spinning disk laser confocal scan head (PerkinElmer UltraView LCI). Dyes were excited at 568 nm with an Ar/Kr laser. Emitted light was captured through 600 nm interference filters by a cooled CCD camera (Hamamatsu OrcaER). Excitation and emission were controlled by a Lambda 10-2 filter wheel and controller (Sutter Instrument). Images were acquired once per minute and analyzed with NIS-Elements AR software (Nikon). After collecting baseline data for 5 min, release was stimulated by applying 50 mM KCl saline (in mM: 68.6 NaCl, 50 KCl, 1.8 CaCl₂, 0.5 MgCl₂, 5 glucose, and 10 HEPES, pH 7.8) for another 15 min.

Wen X., Saltzgaber G.W, & Thoreson W.B. (2017). Kiss-and-Run Is a Significant Contributor to Synaptic Exocytosis and Endocytosis in Photoreceptors. Frontiers in Cellular Neuroscience, 11, 286.

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Multimodal Imaging of Cellular Localization

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For protein localization studies, live cells were imaged. For visualization of DNA, cells were fixed with ice cold 70% ethanol. After washing with PBS the fixed cells were incubated with PBS containing 5 mg/ml methyl blue (MB) for 15 min on ice. MB-stained cells were then pelleted by centrifugation and re-suspended in PBS prior to imaging. Cells were mixed with 4′,6-diamidino-2-phenylindole (DAPI) before imaging. One of the two following microscopes were used to collect images: a spinning-disk confocal microscope (Ultraview LCI; PerkinElmer) with a 100× NA 1.40 Plan-Apochromat oil immersion objective and a 488-nm argon ion laser (GFP) or a 594-nm helium neon laser (mCherry and RFP) or a personal microscope system (DeltaVision; Applied Precision) including a microscope (IX71; Olympus), 60× NA 1.42 PlanApo and 100× NA 1.40 UPlanSApo objectives, fixed- and live-cell filter wheels, a camera (CoolSnap HQ2; Photometrics), and softWoRx imaging software. Images on the confocal were captured on a charge-coupled device camera (Orca-ER; Hamamatsu Photonics. Images were manipulated in Image J and imported into Adobe Illustrator for construction of final figures.

Chen J.S., Beckley J.R., Ren L., Feoktistova A., Jensen M.A., Rhind N, & Gould K.L. (2016). Discovery of genes involved in mitosis, cell division, cell wall integrity and chromosome segregation through construction of Schizosaccharomyces pombe deletion strains. Yeast (Chichester, England), 33(9), 507-517.

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