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High Content Screening (La Jolla)
OverviewThe high-content screening (HCS) core facility of the Conrad Prebys Center for Chemical Genomics (Prebys Center) is part of Sanford-Burnham’s screening center. As part of the Prebys Center, the HCS resource has access to the HTS plate and liquid handling infrastructure of the screening center as well as the screening center’s cell culture facility. For details on the other core facilities of the Prebys Center, please refer to the “Links to related Sanford-Burnham core services” section.
The HCS core facility provides assay development, screening, and data analysis/mining expertise and services for high content screens, where the readout is based on images obtained using high-throughput microscopy systems. For assay development and screening of non-imaging assays refer to Assay Development and High-Throughput Screening (La Jolla) or Ultra-High Throughput Screening (Lake Nona).The HCS team has expertise in all areas of high content image-based screens including sample preparation, image acquisition, image analysis, image data management, and algorithm development.
The HCS core has experience conducting phenotypic assays, ranging from functional assays, such as inhibition of phagocytosis and quantification of cytoskeletal changes, to transcriptional reporter modulation, such as activation of insulin promoter activity. HCS projects range from screening of several hundred chemical compounds to libraries of over 300,000 compounds. Additionally, the Prebys Center screening center is co-located with the Sanford-Burnham’s Functional Genomics (FGC) core, fostering collaboration between the HCS and FGC resources for RNAi screening projects.
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Tech - Shared Resources: CPCCG QuickJump 4 Items
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Services
The HCS core facility's experienced staff is available to help investigators design and execute image-based assays by providing the following services:
Conceptualization and design of image-based assays for medium- and high-throughput screening.
For small scale screens:
- training/use of HCS instruments
- training/use of analysis software
- performing selection, development, and/or validation of image anaylsis methods and/or assay read-outs
For large scale screens:
- optimizing assay biology
- developing algorithms and read-outs
- performing assay miniaturization/validation
- screening in collaboration with HTS or Functional Genomics facilities
Aid in image data management, HC data analysis and visualization
HCS Project Examples
Image-based high content assays are usually employed when an assay utilizes multiplexed fluorescent labels, investigates sub cellular localization or spatial distribution of fluorescence. The technique can also be used when evaluating morphological changes of cells or cellular structures such as shape or size, quantifying co-localization of multiple labels, or monitoring the response of a sub-population of cells. Because of the high content nature of image-based assays, the multi-parametric assay read-outs can provide additional biological or disease relevant information. Some examples of medium and high throughput screening campaigns utilizing image-based high content assays are provided in the table below.
Example HCS Campaigns Executed at Sanford-Burnham's Prebys Center
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Assay
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Cellular Function
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HCS Analysis
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HCS Library & Format
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Bioparticle Uptake Inhibition*
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Phagocytosis
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Particle Count
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110k (chem)/384-well
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Beta-Catenin Activation*
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Signaling
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(Trans-) Location
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200k (chem)/384-well
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Invadopodia
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Fluor. Aggregates/
Structures
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50k (chem)/384-well
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Cilia Modulators
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Cytoskeleton,
Developmental
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Fluor. Aggregates/
Structures
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50k (RNAi)/384-well
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Insulin Promoter
Modulation*
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Signaling, Gene/Protein Expression
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Fluor. Intensity/Sub-population
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225k (chem)/384-well
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PML-Oncogenic
Domain
Activation
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Nuclear Foci
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Fluor. Aggregates
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380k (chem)/384-well
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Lipid Droplet Inhibition*
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Metabolism, Storage
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Fluor. Intensity/Fluor. Aggregates
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300k (chem)/384-well
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GPCRs (Transfluor™ β-arrestin)*
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Signaling
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Fluor. Aggregates/Spatial Distribution
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3 x 300k (chem)/384-well
1 x 330k (chem)/1536-well
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* MLPCN project, refer to PubChem.
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Equipment and Resources
Liquid Handling
Robotic liquid and plate handling equipment and resources are used in common by all groups within the CPCCG and are described in detail under High Throughput Screening (La Jolla) and Ultra-High Throughput Screening (Lake Nona). For information on available screening libraries, refer to Compound Management for chemical libraries and Functional Genomics for RNAi libraries.
HCS Imaging Systems
The HCS core facility contains state-of-the-art automated high-throughput microscopy instruments. Depending on the instrument, autofocusing is either image-based (IC200, Eidaq100) or laser-based (IN Cell 1000, Opera QEHS, Celigo). These instruments can be used with different objectives ranging from 4x to 60x magnification and have the ability to image standard clear-bottom multi-well plates (96, 384, 1536), preferably black plates with optical quality glass or plastic bottoms with a bottom thickness of ~200um. The INCell1000 and Opera QEHS in La Jolla have automatic plate loading capabilities (>40 plates), while the Opera QEHS in Lake Nona is fully integrated into the three-arm robotic uHTS system. For a summary of each HCS instrument’s capabilities refer to the table below.
HCS System Capabilities
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System:
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Objectives:
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Live Cell:
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Confocal:
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Slides:
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Bright-
field:
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Wavelengths (or equivalent):
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Celigo
(La Jolla)
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3.5x
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No
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No
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No
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Yes
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DAPI/FITC/Cy3
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INCell1000
(La Jolla)
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10x, 20x ELWD (air)
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No
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No
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No
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No
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DAPI/FITC/Cy3;
FITC/Cy5;
DAPI/Cy5; FITC
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Eidaq100
(La Jolla)
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10x–40x (air)
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No
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No
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Yes
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Yes
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DAPI/FITC/Cy3;
DAPI/FITC/Cy3/Cy5
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IC200 (Q2‘12)
(La Jolla)
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10x–40x (air)
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Yes
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No
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No
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No
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DAPI/FITC/Texas-Red/Cy5
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Opera QEHS
(La Jolla)
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10x–40x ELWD (air),
20x–60x (H2O)
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No
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Yes
(Spin. Disk)
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Yes
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No
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DAPI/Cy3/Cy5;
DAPI/FITC/Cy5;
DAPI/FITC/Cy3/Cy5
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Opera QEHS
(Lake Nona)
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4x–40x ELWD (air),
20x, 40x (H2O)
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No
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Yes
(Spin. Disk)
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No
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Yes
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DAPI/Cy3/Cy5;
DAPI/FITC/Cy5;
DAPI/FITC/Cy3/Cy5
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HCS Image Analysis and Image Data Management Software
A variety of image processing, data analysis, and visualization tools are available, including InCell Investigator (GE), CytoShop (Q3DM/Beckman Coulter), CyteSeer (Vala Sciences), Acapella (PE), Matlab (The Mathworks), Photoshop (Adobe), ImageJ (NIH), CellProfiler/CellVisualizer (nonprofit open source), and Volocity 3D/time course analysis software (PE). For screen data analysis and mining, CBIS (ChemInnovation) screening database software, as well as Spotfire (Tibco), Genedata Screener, and Prism (Graphpad) data analysis software are available. With these software packages the HCS team can rapidly apply available HCS solutions and algorithms, as well as develop custom HCS solutions, algorithms and tools for the wide variety of HCS projects.
To streamline HCS processing, visualization, and data management, the HCS team implemented a local storage system (LSS) dedicated to HCS based on the Columbus image-data management system (PE). The Columbus system provides ~12TB of fast access storage at the La Jolla site and handles image and data file management for currently ongoing HCS projects. A similar LSS has been established at the Orlando site. The LSSs at both sites are connected via high-speed Sanford-Burnham intranet and provide scientists with web-based access to image visualization, analysis, and data visualization tools. The Columbus image-data management system provides web-based upload capabilities for Opera, INCell 1000, IC200, Eidaq100, and Celigo instruments. It is integrated with the Volocity 3D/time course visualization and analysis package, and is also directly linked to Genedata Screener software for a complete HCS image-data analysis and visualization solution.
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A cell-based high-content screening assay reveals activators and inhibitors of cancer cell invasion.
Quintavalle M, Elia L, Price JH, Heynen-Genel S, Courtneidge SA.
Science Signaling 4(183):ra49, 2011.
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Identification of the GPR55 agonist binding site using a novel set of high-potency GPR55 selective ligands.
Kotsikorou E, Madrigal K, Hurst D, Sharir H, Lynch D, Heynen-Genel S, Milan L, Chung T, Seltzman H, Bai Y, Caron M, Barak L, Abood M, Reggio P.
Biochemistry 50(25):5633-47, 2011.
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A high-content screening (HCS) assay for the identification of chemical inducers of PML oncogenic domains (PODs).
Yip KW, Cuddy M, Pinilla C, Giulanotti M, Heynen-Genel S, Matsuzawa S, Reed JC.
Journal of Biomolecular Screening 16(2):251-8, 2011.
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Targeting of the orphan receptor GPR35 by pamoic acid: a potent activator of extracellular signal-regulated kinase and β-arrestin2 with antinociceptive activity.
Zhao P, Sharir H, Kapur A, Cowan A, Geller EB, Adler MW, Seltzman HH, Reggoi PH, Heynen-Genel S, Sauer M, Chung TD, Bai Y, Chen W, Caron MG, Barak LS, Abood ME
Molecular Pharmacology 78: 560-68, 2010, PMID: 20826425.
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Functional genomic screen for modulators of ciliogenesis and cilium length.
Kim J, Lee JE, Heynen-Genel S, Suyama E, Ono K, Lee KY, Ideker T, Aza-Blac P, Gleeson JG.
Nature 464(7291): 1048-51, 2010.
Read More
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Tech - Shared Resources: Prebys Center Links Related
Links to Related Sanford-Burnham Core Services
The Conrad Prebys Center of Chemical Genomics provides a comprehensive suite of capabilities
for preclinical small molecule drug discovery by providing specialized expertise within our eight core facilities:
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