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High-Throughput Screening Core (Penn)

Directors: Cherry, Sara, Ph.D.; Schultz, David C., Ph.D.

Summary:

The High-throughput Screening (HTS) core provides the Perelman School of Medicine community with professional HTS screening services to identify genes or organic small molecule modulators of signaling pathways, cellular phenotypes, and protein function in models of human disease. Core staff will educate and assist scientists with HTS assay development, optimization, miniaturization, and validation; maintain libraries of siRNA, shRNA, cDNA, and FDA approved/FDA-like organic small molecule libraries for HTS; and provide robotics infrastructure and technically trained staff for HTS, including small screens of user defined libraries. The High-throughput Screening core also provides direct assistance with preparation of grant applications by drafting experimental designs approaches and providing Letters of Support, offering HTS resources and analysis expertise for the proposed research.

Affiliations:

People:

Resources:

Instruments

  • Biotek ELx405 microplate washer ( Microplate washer )

  • BSL2 tissue culture capability ( Environmental chamber )

    biosafety level 2 tissue culture

  • Bulk reagent dispensors ( Liquid handler )

  • EnVision Xcite multi-mode microplate reader ( Microplate reader )

    The PerkinElmer EnVision Xcite is a multi-mode plate reader
    capable of measuring standard detection labels in 96-, 384-, and 1536-well plates, including absorbance, fluorescence intensity, luminescence, ultra-luminescence, time-resolved fluorescence, fluorescence polarization, and AlphaScreen (Amplified Luminescence Proximity Homogeneous Assay).

  • FLIPR Tetra screening system ( Microplate reader )

    Measurement Technology:
    • Integrates liquid handling with rapid whole plate imaging of
    fluorescence and luminescence
    • Can read at sub-second intervals, which enables the kinetics of
    the response to be captured
    • Integrated pipettor enables successive liquid additions,
    providing an opportunity to detect agonists, antagonists, and
    allosteric modulators all in one assay

    Assays:
    1. Intracellular Ca2+ flux: cell permeable Ca2+ sensitive fluorescent dye (e.g. cardiotoxicity);
    2. Fatty acid or neurotransmitter uptake of fluorescence dyes.
    3. Membrane potential: lipophilic, anionic, fluorescent dye that partitions across the cytoplasmic membrane of live cells based membrane potential.

  • ImageXpress Micro XLS ( Digital microscope )

    Enables automated acquisition of multi-channel
    fluorescence images in microtiter plates (5 colors)

    Phenotypic assays up to 40X
    • Physical properties of cells (size, shape, etc.)
    • Puncta formation (autophagy, lipid droplets, etc)
    • Activity (migration, invasion, neurite outgrowth)
    • Expression and localization of native proteins
    • Measurement of fluorescent labels (antibody stains,
    EdU/BrdU, Calcein, PI uptake, mitochondria, etc.) • FRET

  • Matrix Wellmate ( Microplate dispenser )

    Matrix Wellmates with integrated stackers and a ThermoFisher Multidrop combi for bulk reagent dispensing. These instruments can be programmed to fill specific plate columns as desired by the user, and is capable of dispensing 0.5-2500 μl per well. The

  • Perkin Elmer Janus Modular Dispensing Tool automated pipetting station ( Liquid handler )

    with 96/384 Modular Dispensing Tool (MDT) and Verispan 8-tip for automated liquid pipetting

Reagents

  • Diversity library ( Chemical library )

    "Chembridge (32,000 total): 20,000 from Core set and 12,000 from Express Pick set
    ChemDiv (12,000 total): SMART library"

    "• Started with ~200,000 compounds
    • Used modified Lipinski parameters to filter the set.
    • Removed Reactive groups (e.g. Michael acceptors) and compounds with undesirable functionalities.
    • Duplicates were removed.
    • Lead Finder Clustering and MACCS fingerprints were generated.
    • PAINS1, PAINS2 and PAINS3 substructure filters were applied.
    • REOS filters were applied.
    • Property Based Selection of 50K cpds was performed
    • Vendor re-supply validated"

  • LOPAC ( Known bioactives collection chemical library )

    "Features include:
    • 1,280 pharmacologically active compounds
    • Marketed drugs and pharmaceutically relevant structures annotated with biological activities"

  • MGC cDNA collection (CMV-driven) ( cDNA library )

    • 18,000 full length, sequenced, mouse and human cDNAs pre-cloned into pCMV-SPORT6
    • Insert is fully sequenced and guaranteed to match corresponding BC Accession Number
    • Expression-ready vectors eliminate additional cloning steps
    • Robust CMV promoters drive cDNA expression
    • Gateway sites flanking coding sequence allow for additional flexibility
    • Stored as bacterial glycerol stocks, arrayed in 96 well microtiter plates

  • MicroSource Purified Natural Products library ( Natural product extracts chemical library )

    "The NatProd Collection provides 800 pure natural products and their derivatives, which include simple and complex oxygen heterocycles, alkaloids, sequiterpenes, diterpenes, pentacyclic triterpenes, sterols, many other diverse representatives. The collection is available in 96 well microplates as10mM DMSO solutions or as 5mg solid samples in glass vials."

  • Non-coding RNAs ( siRNA library )

    Screen a library of siRNAs against
    • lncRNAs (human, 2220 targets)
    • miRNA mimics/antagonists (human V20, 25550 targets each)

  • Selleck Chemicals Bioactive compound library ( Known bioactives collection chemical library )

    Kinase Inhibitors, Epigenetic Inhibitors, Cancer compounds, GPCR/Ion Channel, Metabolism, Microbiology, FDA approved/FDA-like

  • shRNA library ( shRNA library )

    Human TRC 2.0 and Mouse TRC1.0 Lentivirus shRNA library
    • Screening pools: GO categories; user-defined sets
    • Order groups/individuals

  • siRNA genetic library ( siRNA library )

    • human genome-wide
    • human GO categories
    • user-defined human and mouse

Services

  • Assay development service ( Support service )

    Biochemical, cell and high-content assay development is available.

    Assay development involves consultation, technology assessment, assay design, optimization, and miniaturization.

  • Custom siRNA library service ( Material production service )

    "• Custom siRNA libraries are provided by Ambion/Life
    technologies
    • Minimum order of 20 siRNAs REQUIRED
    • 0.1 nmol of Silencer Select siRNA (human)
    • 1 nmol of Silencer siRNAs (human and mouse)
    • siRNAs are received in microtiter plates (96 or 384)
    • All orders are submitted through the HTSC"

  • High-throughput screening data analysis ( Data analysis service )

    • Normalization, annotation
    • HCA analysis sequence dev.
    • Screen reports

  • High-throughput screening equipment training ( Training service )

  • High-throughput screening service ( Material analysis service )

    High-throughput screening:
    • pharmacologically active compounds
    • diversity collections
    • focused libraries (e.g. annotated inhibitors)
    • siRNA, cDNA, and shRNA
    • library plates

  • HTS Core equipment access ( Access service )

    with or without help

  • HTS grant preparation support service ( Support service )

    • letters of support
    • experimental design section

  • shRNA library services ( Service )

    "Distribution of Individual clones
    • $100.00 per Gene Target (5+ clones)
    • Investigator should sequence confirm shRNA sequence
    • Investigator responsible for validating knock-down
    Preparation of Custom Gene Sets
    • Cherry-pick and array clones of interest into new plates ‒ GO categories (e.g. Epigenetic targets) ‒ User-defined sets (e.g. ‘omics validation)
    • Glycerol stock plate + pooled plasmid DNA
    • 150 gene Targets/625 clones: $2000
    • 600 gene Targets/3000 clones: $5000"

  • Small-scale screening service ( Material analysis service )

    Small-scale screening:
    • user-defined sets of genes
    • user-defined cell-types across small libraries of bioactive compounds/inhibitors
    • synergy screening with smaller libraries
    • library plates (e.g. kinome)

Software

  • MetaXpress ( Software )

    • Easy analysis pipelines in-house
    • Computational analysis of images enables measurement of multiple cellular properties of interest at the object/cell level and/or population level.

  • OpenHTS ( Software )


Web Links:

Last updated: 2016-04-26T13:56:24.822-04:00

Copyright © 2016 by the President and Fellows of Harvard College
The eagle-i Consortium is supported by NIH Grant #5U24RR029825-02 / Copyright 2016