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Ultraview ers6

Manufactured by PerkinElmer
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The Ultraview ERS6 is a high-speed confocal microscope system designed for live-cell imaging and analysis. It features a compact and modular design, allowing for easy integration into various research environments. The system provides high-resolution, multicolor imaging capabilities, enabling users to visualize and study dynamic cellular processes in real-time.

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Lab products found in correlation

Axiovert 200m, by Zeiss (6 mentions) Orca flash 4.0 camera, by Hamamatsu Photonics (2 mentions) Fluorescent phalloidin, by Thermo Fisher Scientific (1 mentions) Leibovitz s l 15 media, by Thermo Fisher Scientific (1 mentions) Plan apo 1.4na, by Zeiss (1 mentions) Plan apochromat 1.4 na objective, by Zeiss (1 mentions) Orca er camera, by Hamamatsu Photonics (1 mentions) Volocity software, by PerkinElmer (1 mentions)

4 protocols using ultraview ers6

1

Imaging F-Actin Dynamics in Activated T Cells

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Activated CD4+ T cells were resuspended in Leibovitz’s L-15 media (Gibco) supplemented with 2 mg/mL glucose and incubated at 37° C for 20 min. T cells were then added to ICAM-1 coated coverslips (coated with 2 μg/mL overnight at 4° C). After 20 min at 37° C, cells were washed in L-15 and fixed in 3.7% paraformaldehyde in PBS. Cells were then blocked and permeabilized in PSG (PBS, 0.01% saponin, 0.05% fish skin gelatin) for 20 min, followed by 45 min incubation with fluorescent phalloidin (Molecular Probes) in PSG. Cells were washed in PSG, mounted, and imaged using a 63× PlanApo 1.4 NA objective on an Axiovert 200M (Zeiss) with a spinning disk confocal system (Ultraview ERS6; PerkinElmer). Four z-planes spanning a total of 0.75 μm were collected at the cell-surface interface with an Orca Flash 4.0 camera (Hamamatsu). Image analysis of the rendered stacks was conducted using Velocity v6.3 software. Cells were identified using the “Find Objects” command, using a low threshold on the actin channel, and total phalloidin staining was quantified per cell based on integrated pixel intensity.
Roy N.H., Mammadli M., Burkhardt J.K, & Karimi M. (2020). CrkL is required for donor T cell migration to GvHD target organs. Oncotarget, 11(17), 1505-1514.
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2

High-Resolution Imaging of ER Stress

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HAP1 cells were plated on 35-mm microscopy-grade plastic dishes (Ibidi). After dox induction and subsequent imposition of chemical ER stress, the cells were imaged over 8 h using a Marianas fluorescence microscope equipped with an OKO Lab CO2 enclosure on a Zeiss inverted platform, with a 63X Plan-Apochromat 1.4 NA objective. Images were collected as in (23 (link)). Exposure times varied between 0.1 and 0.5 s, depending on sample intensity, unless otherwise specified. In some experiments, cells were imaged using a 63X Plan Apo 1.4 NA objective on an Axiovert 200M (Zeiss) with a spinning disc confocal system (UltraVIEW ERS6, PerkinElmer). Images were collected using an ORCA Flash 4.0 camera (Hamamatsu Photonics) using Volocity V.6.3.1 acquisition software.
Ricci D., Tutton S., Marrocco I., Ying M., Blumenthal D., Eletto D., Vargas J., Boyle S., Fazelinia H., Qian L., Suresh K., Taylor D., Paton J.C., Paton A.W., Tang C.H., Hu C.C., Radhakrishnan R., Gidalevitz T, & Argon Y. (2021). An interdomain helix in IRE1α mediates the conformational change required for the sensor's activation. The Journal of Biological Chemistry, 296, 100781.
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3

Quantifying CAR T-cell-Target Conjugation

Cited 1 time
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Immune conjugates were generated as previously described39 (link). Briefly, CAR T cells were stained with CellTrace Violet (CTV) and were combined with GFP+ Nalm6 cells at an effector:target ratio of 2:1. Co-cultures were incubated for either 5 or 15 minutes at 37°C, fixed and conjugate formation (CTV+GFP+ doublets) was quantified by flow cytometry.
For analysis of CAR distribution on the cell membrane, GFP tagged lentiviral constructs were generated by introducing in-frame fusions of GFP at the 3’ end of the CAR construct using standard molecular cloning. T cells were spun at 500rpm for 3 minutes, followed by a 10 minute incubation at 37°C. Cells were then applied to poly-L-lysine coated glass coverslips, washed and fixed with 4% paraformaldehyde. Coverslips were mounted and imaged using a 63x PlanApo 1.4 NA objective on an Axiovert 200M (Zeiss) with a spinning disk confocal system (Ultraview ERS6; PerkinElmer). Z-planes were collected every 0.25 μm, spanning a total of 10 μm with an Orca Flas 4.0 CMOS camera (Hamamatsu). Analysis was done using Volocity v6.3 software and ImageJ open source software was used to prepare representative images.
Singh N., Frey N.V., Engels B., Barrett D.M., Shestova O., Ravikumar P., Cummins K.D., Lee Y.G., Pajarillo R., Chun I., Shyu A., Highfill S.L., Price A., Zhao L., Peng L., Granda B., Ramones M., Maggie Lu X., Christian D.A., Perazzelli J., Lacey S.F., Roy N.H., Burkhardt J.K., Colomb F., Damra M., Abdel-Mohsen M., Liu T., Liu D., Standley D.M., Young R.M., Brogdon J.L., Grupp S.A., June C.H., Maude S.L., Gill S, & Ruella M. (2021). Antigen-independent activation enhances the efficacy of 41BB co-stimulated CD22 CAR T cells. Nature medicine, 27(5), 842-850.
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4

Quantifying MTOC Polarization in NK-Target Cell Conjugates

Cited 1 time
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Immunofluorescence microscopy was carried out essentially as described previously (15 (link)). YAC-1 cells were labeled with CMAC, mixed 1:2 with NK cells, and conjugation was induced by centrifugation for 5 minutes. After an additional 10 minutes, cells were gently resuspended and incubated on Poly-L-lysine coated coverslips for 10 minutes. Cells were then fixed with 3% paraformaldehyde, permeabilized, and labeled with primary antibodies and fluorescent secondary antibodies. Conjugates were imaged using a 63× PlanApo 1.4 NA objective on a spinning disk confocal system (UltraView ERS 6; Perkin Elmer, Waltham MA), equipped with an ORCA-ER camera (Hamamatsu Photonics, Bridgewater NJ) and Volocity software (v6.1.1; PerkinElmer). MTOC to synapse measurements were performed manually as follows: The border of the YAC-1 cell was determined based on CMAC fluorescence intensity, and the center of the MTOC was defined as the pixel with the brightest intensity in the 488 channel (anti-tubulin). The distance from the MTOC center to the nearest site on the YAC-1 border was then measured using Volocity software. Image preparation was performed with ImageJ.
Freund-Brown J., Choa R., Singh B.K., Robertson T.F., Ferry G.M., Viver E., Bassiri H., Burkhardt J.K, & Kambayashi T. (2017). Murine natural killer cells degranulate and retain cytotoxic function without store-operated calcium entry. Journal of immunology (Baltimore, Md. : 1950).
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