U-2-OS cells were grown on clear bottom 96-well plates (Greiner) and either pre-extracted with cold 0.5% Triton X-100 CSK buffer for 5 min before fixation or directly fixed in 4% formaldehyde for 10 min. For EdU (5-ethynyl-2′-deoxyuridine) labeling, cells were incubated with 40 μM EdU for 20–30 min. EdU was detected using Click-iT Plus EdU Kit for Imaging (C10640). Plates were imaged on a Perkin Elmer Operetta high-content imaging system or Olympus Scan-R imaging system using a 20x objective. 35 fields per well were imaged, and ∼2,000 cells per condition were analyzed. Single-cell fluorophore intensities were extracted using the Columbus system (Perkin Elmer) or Scan-R analysis software. Cell cycle phases were gated based on DAPI and EdU or PCNA intensities. Graphs were generated using Tableau 2019.3 and GraphPad Prism.9 software.
Scanr imaging system
Manufactured by Olympus
The ScanR imaging system is a high-performance automated microscopy solution designed for cell-based assays and high-content screening. It provides advanced imaging capabilities and automated image acquisition and analysis to support a wide range of life science applications.
Automatically generated - may contain errors
Lab products found in correlation
Operetta high content imaging system, by Olympus (1 mentions)
Columbus system, by PerkinElmer (1 mentions)
Clear bottom 96 well plates, by Greiner (1 mentions)
Ix83 microscope, by Olympus (1 mentions)
Orca flash4.0 v2 scmos camera, by Hamamatsu Photonics (1 mentions)
Axiocam mrm camera, by Zeiss (1 mentions)
200m inverted microscope, by Zeiss (1 mentions)
Axiovision software, by Zeiss (1 mentions)
Hamamatsu orca er camera, by Hamamatsu Photonics (1 mentions)
Metamorph software version 6.2r6, by Molecular Devices (1 mentions)
4 protocols using scanr imaging system
1
High-Content Imaging of Cell Cycle
2
Multi-Wavelength Imaging with Olympus ScanR
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The Olympus ScanR imaging system based on an inverted motorized Olympus IX83 microscope was equipped with one set of bandpass filters for multi‐wavelength acquisition: DAPI (ex BP 395/25, em BP 435/26), FITC (ex BP 470/24, em BP 511/23), TRITC (ex BP 550/15, BP 595/40) and Cy5 (ex BP 640/30, em BP 705/72). The associated objectives used were a 20× NA 0.75 UPLSAPO and a 40× NA 0.90 UPLSAPO air objective. Images were acquired with 12‐bit dynamics on a 16‐bit 2,048 × 2,048 pixel Hamamatsu ORCA‐FLASH 4.0 V2 sCMOS camera with pixel size of 6.5 μm.
3
Fluorescence In Situ Hybridization Analysis
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The miRNA-transfected HCT-116 cells were trypsinised and swelled for 15 min using 0.075 M KCl hypotonic solution. The cells were fixed using methanol/acetic acid (3 : 1) for 1 h at 4 °C. The fixed cells were dropped on clean glass slides and incubated at RT for 10 min. Vysis LSI ETV6(TEL)/RUNX1(AML1) ES Dual colour probe and Vysis LSI 13(13q14) SpectrunGreen probe were purchased from Abbott Inc., Abbott Park, IL, USA. The samples were hybridised according to the manufacturer's instructions. The Image acquisition and analysis was done using ScanR Imaging system (Olympus Corporation). The FITC and Cy3 channels were used to detect green and red signals respectively.
4
Quantifying Kinetochore Dynamics in Fixed Cells
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Fixed cells were imaged using a Zeiss inverted 200M microscope (Zeiss GmbH, Jena, Germany) equipped with Hamamatsu ORCA-ER camera (Hamamatsu Photonics, Hamamatsu City, Japan), and Metamorph software version 6.2r6 (Molecular Devices, Downingtown, PA, USA). Kinetochore intensities were quantified from maximum projections created from a Z-stack of images acquired with 0.3–0.5 μm step interval. For FISH and analysis of nuclear morphology, ScanR imaging system (Olympus Corporation) was used. Live cell imaging was conducted using IncuCyte live-cell imager (Essen Instruments Ltd., Hertfordshire, UK) or a Zeiss inverted 200M microscope (Zeiss GmbH) equipped with an environment chamber, AxioCam MRm camera and AxioVision software (Zeiss GmbH).
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