University of Pittsburgh Cancer Institute (UPCI)

CCSG Acknowledgement

Required CCSG Acknowledgement

The NCI requires that publications acknowledge the UPCI CCSG support, and they are tracking compliance. If a UPCI CCSG-supported Shared Resource provided data used in your publication, please include the following statement in the acknowledgment section of your publication(s):

"This project used the UPCI [insert name(s) of shared resource(s)] that [is/are] supported in part by award P30CA047904."

Shared Resource Directors: Please make sure to include this statement on all of your order forms, contracts, etc. as a reminder to your users to acknowledge the UPCI CCSG support.


Imaging Cytometry

The Imaging Cytometry platform houses the Cellomics ArrayScan VTI cytometer, which enables the collection of quantitative data on many cellular processes, including:

• Receptor activation
• Cell membrane receptor binding
• GPCR internalization
• Labeled ligand internalization
• Cell proliferation
• Cell morphology
• Cell survival signaling
• Cell migration signaling
• Toxicity
• Cell Viability
• Apoptosis/Necrosis

• Nuclear count
• Fluorescent protein localization
• Transcription factors
• Reporter gene expression
• Cell cycle status
• DNA replication studies
• Nuclear-cytoplasmic translocation
• Plasma membrane translocation
• Neurite outgrowth
• Neuronal profiling
• Tube formation
• Microtubule arrangement
• Cytoskeletal reorganization
• Micronuclei formation
• Genotoxicity
• Hepatotoxicity
• Oxidative stress
• Phospholipidosis
• Cholestasis
• Calcium homeostasis
• Neurotoxicity
• Stress response
• Subpopulation analysis

Advantages of Imaging Flow Cytometry over Conventional Flow Cytometry

  • Ability to localize fluorescence within cells and quantitatively determine fluorescence in defined cellular features (e.g., nucleus versus cytoplasm, endosomes versus lysosomes)
  • Ability to co-localize fluorescent signals in the entire cell or within selected cellular features (e.g., quantifying tagged proteins within organelles)
  • Ability to localize fluorescent signals during cellular interactions (e.g., adhesion molecules in the immune synapse)
  • Availability of 35 intensity-based and morphologic parameters per channel with ability to create user-defined parameters based on Boolean and algebraic calculations (e.g., nucleus-to-cytoplasm ratio)
  • Ability to view images (single or composite fluorescent images, or brightfield) associated with a single point or a region on a bivariate scatter plot
  • Morphologic parameters can serve as surrogates for one or more fluorescent parameters, freeing up fluorescent channels (e.g., detection of apoptosis cells by comparing nuclear area and peak nuclear pixel intensity, ability to distinguish prophase, metaphase and anaphase on the basis of nuclear texture, size and aspect ratio)

Advantages over Fluorescence Microscopy-Based Image Analysis

  • Higher throughput by 2-3 orders of magnitude gives better statistical analysis
  • Choice of objects and subpopulations for analysis is objective rather than subjective

Advantages over Fluorescence Activated Cell Sorting Followed by Image Analysis

  • Can “sort” on calculated morphologic features and by location of fluorescence within a cell
  • Does not require two complex instruments which are usually not in the same location (1.8 miles apart in our case)
  • No loss of cells or viability issues
  • Can perform kinetic experiments

Advantages over Image Scanning Cytometry

  • Better suited to cells in suspension (simplified sample handling)
  • Gives light scatter measurements
  • Findings more easily translated to flow cytometry (e.g., for polychromatic multiparameter analysis or cell sorting)
  • Cells in suspension are not altered in function or appearance by adherence to a solid substrate

Instrumentation

Cellomics ArrayScan VTI Imaging Cytometer

The Cellomics ArrayScan VTI shown in current configuration with automatic robotic plate loading capability The Imaging Cytometry platform centers on a Cellomics ArrayScan VTI imaging cytometer with up to 40X magnification and a 120W mercury-halide white light source capable of excitation from 300nm-700nm and up to 5-color capability. Related equipment also includes a Twister robotic plate loader, a Zeiss Apotome for pseudo-confocal capability (7uM sectioning), a 1T server for data storage and a dedicated data analysis terminal.

The ArrayScan VTI is an automated fluorescence image analysis system with integrated image analysis and data management that together allow the user to perform High Content Screening analysis (HCS). Conventional inverted fluorescence microscopy is applied with robotics to enable fully automated imaging of cells in multi-well cell culture plates. The VTI brings three common technologies--a fluorescent plate reader, fluorescent microscope, and flow cytometer--into a single instrument. The focus of the VTI is to establish event dynamics across populations and subpopulations like a flow cytometer, while incorporating sophisticated image processing to allow for spatial discrimination of fluorescent dyes within the cell. Sophisticated image analysis enables quantification of cellular fluorescence on a large scale to include highly complex phenomenon like morphological changes involving sub-cellular constituents, whole cell morphology, and multi-cellular structures, for example. Biological content is further increased through spatial analyses of targets labeled with fluorescent dyes. The integrated database allows for remote sharing of data and images and features logical in-line operations like automated dose response curve reporting and relationships within reams of cellular data.

More information about the ArrayScan VTI

Some commonly used fluorophores compatible with the ArrayScan VTI:

• Hoechst 33342 • YOYO-1 • Rhodamine • Texas Red • DRAQ5
• DAPI • Alexa 488 • Cy3 • Alexa 568/594 • Alexa 647/680
• FITC • Calcein • Alexa 546 • LsyoTracker Red • TOTO3
• GFP, EGFP • BODIPY-FL • DyLight 549 • MitoTracker Red