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High content screening workflow - PowerPoint PPT Presentation


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High content screening workflow. HTS (High throughput screening): Biochemical or cell based; mostly end-point assays HCS (High content screening): Cell based; fluorescence imaging; kinetic output / live cell imaging. High Content Screening (HCS)  applications :

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slide1

High content screening workflow

HTS (High throughput screening): Biochemical or cell based; mostly end-point assays

HCS (High content screening): Cell based; fluorescence imaging; kinetic output / live cell imaging

slide2

High Content Screening (HCS)  applications:

  • Target identification - Genome-wide functional siRNA screening.
  • Primary screening - Screening of libraries of compounds with meaningful functional cellular readout.
  • Secondary screening / follow up testing - Dose responses for active compounds, complementary assay to identify false positives. Time courses of the assay in live cells.
  • Safety assessment: In vitro tests using organ specific cells (e. g. hepatocytes, neurons) in order to get early information on the side effects of drugs.
  • Systems biology: Genome-wide functional siRNA screening. Recent advances in yeast biology, i. e. the availability of genome-wide collections of gene-knockout mutants, has greatly facilitated yeast-based gene function discovery.
  • Stem cell biology: The search for stem cell based therapies and scalable human primary cell sources for drug discovery have created an enormous need for stem cell reprogramming and differentiation screening methods.
slide5

High content screening platforms and categories

  • Thermo Fisher/Cellomics
      • Cytotoxicity and apoptosis
      • Genotoxicity, DNA damage and repair
      • Inflammation and Cell Stress
      • Cell Signaling and Transcription Factors
      • Cell Cycle and Proliferation
      • Cell Morphology and Phenotype Changes
  • Perkin Elmer
  • GE Healthcare
  • Molecular Devices
  • Genetix
  • BD Biosciences
slide6

Specifications of high content screening analysis

  • Assay features
    • Assay category
    • Assay name
  • Target
    • Biological process
      • Potentiator
    • Pathway/Target
      • Species
    • Cell-line
    • Phenotype
  • Detection
    • Primary antibody
    • Secondary antibody
    • Dye
    • Dye target
  • Image analysis
    • Image analysis module
    • Detection target
    • Detection quantification
    • Endpoint
    • Controls
    • Data normalization
slide7

Example specifications

  • Assay features
    • Assay category: Cytotoxicity and apoptosis
    • Assay name: Apoptosis 1
  • Target
    • Biological process: Apoptosis
      • Potentiator: Doxorubicin
    • Pathway/Target: Mitochondrial pathway of apoptosis
      • Species: Human
    • Cell-line: Hek-293
    • Phenotype: Nuclear DNA condensation
  • Detection
    • Primary antibody: Caspase-3
    • Secondary antibody: Anti-caspase-3 conjugated with Alexa 594
    • Dye: Hoechst
    • Dye target: DNA
  • Image analysis
    • Image analysis module: Cell health profiling bioapplication
    • Detection target: Nuclear DNA
    • Detection quantification: Fluorescence intensity
    • Endpoint: Concentration response; activity based; generic
    • Controls: DMSO negative control
    • Data normalization: Z’-factor
slide8

High content analysis

  • Application
    • Channel 1
      • Cell counts and density
        • Output cell features: e.g. Cell number
        • Output well features: e.g. ValidObjectCount
      • Morphology and location
        • Output cell features: e.g. ObjectArea and Status
        • Output well features: e.g. Mean, SD
      • Intensity
        • Output cell features: e.g. Total intensity and status
        • Output well features: e.g. Mean, SD
    • Channel 2
      • Intensity
        • Output cell features: e.g. Total intensity and status
        • Output cell features: e.g. Mean, SD
      • Morphology
        • Output cell features
        • Output cell features
slide9

Existing Cell line ontologies

Cell line MCCL: http://bioportal.bioontology.org/visualize/39927

Cell line CLKB: http://bioportal.bioontology.org/visualize/40261

ATCC has 3487 and CLDB has 5367 cell lines

Existing fluorophore ontologies

Molecular Imaging and Contrast Agent Database (MICAD): http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=micad

Biological imaging methods: http://www.ebi.ac.uk/ontology-lookup/browse.do?ontName=FBbi

slide10

Cell line MCCL ontology

  • Anatomical entity
  • animal kingdom
  • cell
  •     cell in vivo
  •     experimentally modified cell
    •     cell line cell
      •     permanent cell line cell
        •     1321N1
        •     16610D9
        •     16HBE14o-
        •     1HAEo
        •     2008/C13*5.25
        •   293
        •     2fTGH
        •     308
        •     30A5
        •     32D
        •     32Dcl3
        •     3T3-Swiss albino  
slide12

Molecular Imaging and Contrast Agent Database (MICAD) 

OpticalA15

Alexa Fluor 488

Alexa Fluor 647

Alexa Fluor 680

Alexa Fluor 750

AOI987

Au

BODIPY-FL

BODIPY TMR-X

Cy5

Cy5.5

Cy7

Cypate

DiD

DiR

Fluorescein

Fluoresceinisothiocyanate (FITC)

Green fluorescence protein

Green fluorescence protein (GFP), luciferin

HiLyte Fluor 647

Indocyanine green

IR-783

IR-786

IRDye700DX

IRDye78

IRDye 800CW

Luciferin

Malachite green

NIAD-4

NIR2

Oregon Green 488

Pyropheophorbideα (Pyro)

Quantum dot (QD)

Rhodamine green (RhodG)

RhodamineX

SIDAG

Single-walled carbon nanotubes (SWNTs)

Tetramethyl-6-carboxyrhodamine

VivoTag-S680

VM315