Fluorescent Imaging Solutions for High Content Screening

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Fluorescent Imaging Solutions for High Content
Screening

• Herman Fennema
• Field Application Scientist
• Imaging and Microscopy
• April, 2006

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Molecular Probes Campus, Eugene Oregon
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HCS, HCA, HTI, AI, What are they?

• High Content, Context Screening or Analysis
  (H.C.S.)
• High Content Analysis (H.C.A.)
• High Throughput Imaging (H.T.I.), Automated
  Imaging (A.I.)
• The one constant is that spatial information is
  derived.

HCS Defined. First Proposed in 1986.
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The Cs in HCS- Cell, Content, Context
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Invitrogen HCS Product Overview

• Enabling probes
• HCS CellMask cytoplasmic/nuclear stains
• Organelle markers
• Assays
• HCS LipidTOX phospholipidosis and steatosis kit
• Apoptosis assays
• Other cytotoxicity assays
• GeneBLAzer assays
• Expression tags
• Tetracysteine and TC-FlAsH TC-ReAsH products
• Antibodies
• Alexa Fluor and Qdot conjugated antibodies

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The Cs in HCS- Cell, Content, Context
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Two Sets of Novel HCS Products
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Segmentation Tools- Customer Needs
Utility Current segmentation tools Whats needed?
Segmentation tools Hoechst 33342 (nuclear stain) DRAQ5 Cytoplasmic stain-based segmentation Alternative to DRAQ5
Segmentation is a Key Step in HCS Workflow
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HCS CellMask Stain- Red

• Ex/Em 622/645
• Tunable cytoplasmic/nuclear staining
• Low conc. - comparable level of staining in
  cytoplasm and nucleus
• High conc.- stronger staining in nucleus
• Workflow compatibility
• No wash needed- Last step in the staining and
  should be kept in the buffer during imaging

5 uM
1 uM
Segmentation using 5 uM (on BD Pathway HT)
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HCS CellMask Stain- Blue and Deep Red
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CellMask Stains vs. Hoechst Dyes
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CellMask Stains vs. DRAQ5
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Why CellMask Stains
Choices of HCS CellMask Stains- Multiplexing
Flexibility
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Intro to Cytotoxicity
Cytotoxicity- Multi-Facet Phenomenon
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Cytotoxicity Assays
Wheel of Misfortune
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HCS LipidTOX Kit

• Phospholipidosis
• Intracellular accumulation of phospholipids
• Triggered by cationic amphiphilic drugs
• Steatosis
• Intracellular accumulation of neutral lipids
• Triggered by drugs that affect metabolism of
  fatty acids and neutral lipids
• HCS LipidTOX Kit
• One workflow, two readouts
• A complete assay kit for phospholipidosis and
  steatosis
• With control compounds

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Value for HCS - LipidTox Kit
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HCS LipidTOX Kit Phospholipidosis and Steatosis
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The Illuminated Cell
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Cytoskeleton Tubulin

• T34075 TubulinTracker Green
• A11126 antia-tubulin mouse monoclonal IgG

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Viability Assay
Live Dead, Cytotoxicity Assay (L3224) In live
cells, esterases convert Calcein-AM from
non-fluorescent to green. Dead cells accumulate
Ethidium Homodimer, the membrane impermeable
nucleic acid stain to stain the nucleus red.
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Endoplasmic Reticulum

• E12353 ER-Tracker Blue-White DPX
• E34250 ER-Tracker Red
• E34251 ER-Tracker Green
• S34200 SelectFX Alexa Fluor 488 Endoplasmic
  Reticulum Labeling Kit
• B7449 Brefeldin A, BODIPY 558/568 conjugate
  isomer 1

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Cytoskeleton Actin

• A12379 Alexa Fluor 488 phalloidin
• R415 Rhodamine phalloidin
• A12381 Alexa Fluor 594 phalloidin

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Plasma Membrane

• V22885 Vybrant DiI cell-labeling solution
• V22886 Vybrant DiO cell-labeling solution
• V22887 Vybrant DiD cell-labeling solution
• W11262 Alexa Fluor 594 wheat germ agglutinin
• F35355 FM 1-43FX fixable analog of FM 1-43
  membrane stain

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Live Cell Stains that are fixable (or retainable
stains)
The most Workflow friendly set of dyes!
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Live Cell Stains and Live Cell-Fixable Stains
Staining Alive, Staining Alive
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Live Cell Staining Solutions

• Vybrant Assays for Live Cell Study
• Vybrant Phagocytosis Assay Kit
• Vybrant CFDA SE Cell Tracer Kit
• Vybrant MTT Cell Proliferation Assay Kit
• Vybrant Multidrug Resistance Assay Kit
• Vybrant Cell Adhesion Assay Kit (Calcein AM and
  SYTOX Green)
• Vybrant Cell Lineage Tracing Kit NEW!
• Vybrant Cell Metabolic Assay Kit with
  C12-resazurin
• Vybrant Cytotoxicity Assay Kit G6PD release
  assay NEW!
• Vybrant Alexa Fluor 488 or 555 or 594, Lipid
  Raft Labeling Kit
• Vybrant FAM Poly Caspases Assay Kit for flow
  cytometry
• Vybrant Tubulin Staining Kit for live cells
  100 assays NEW!

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Reagents for Multiple Apoptotic Parameters
Timeframes within Camptothecin / Jurkat Model
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The Cs in HCS- Cell, Content, Context
Less Searching More Content
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More on Qdot Nanocrystals
CdS for UV-blue, CdSe for the bulk of the
visible spectrum, CdTe for the far red and
near-infrared. Qdots are bright Extinction
Coefficient Qdots (.5 to 2 x 106 M-1 cm-1)
Similar to RPE1 x 106. Organic dyes 5-10 x 104.
Quantum yields are high as well and they glow
longer, so Qdots 10 20x brighter. By
2-photon, their action cross section volume is 3
orders of magnitude higher. Excellent labels for
conjugation. Brightness provides high
sensitivity. Ideal for Multiplexing !
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Qdot Nanocrystals Optimal For

• Single line devices, with multiple detectors
  (7-8), FLOW, Imaging, uPlate.
• Light Microscopy to Electron Microscopy
  (Ellisman, Deernick).
• Two Photon, Small Animal In Vivo Imaging (SAIVI).
• Confocal 3 D.
• Arrays? When background comes down.
• Live cell surface interrogation primary
  conjugations.
• Archival Pathology.
• Single molecule detection.
• Sensors?
• FRET DONORS.
• Multiplex RNAi Lipofectamine 2000 Dots
  siRNA.

Quantum Dot Corp. and BioPixel Staff in Eugene now
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The Cs in HCS- Cell, Content, Context
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Zymed Research 1º Antibodies 12 Categories

• Angiogenesis
• Apoptosis
• Cancer Markers
• Cell Adhesion, Cytoskeleton, Matrix
• Cell Junctions
• Cell Cycle, Tumor Suppressors, Oncogenic
  Proteins
• DNA Replication Repair RNA Splicing
  Translation
• Neurobiology
• Phospho-Amino Acid Antibodies
• Signal Transduction (includes receptors, kinases,
  phospho-proteins)
• Transcription Factors
• Ubiquitin/Proteasome Protein Degradation Pathway

Research portfolio focused on cancer, cell
biology/signaling, and neuroscience
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Multiplexing
Alexa Fluor Dyes across the spectrum
Color Selection ? Brightness ?
Photostability
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Amplification and Multiplex FISH
Multiplex detection of RNA expressed (7 genes) in
Drosophila embryos
FISH Tag RNA or DNA kits now optimized and
available
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The Cs in HCS- Cell, Content, Context
Less Searching More Content
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Stealth RNAi for target validation
Stealth RNAi has improved specificity and
stability
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The Cs in HCS- Cell, Content, Context
Less Searching More Content
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GeneBLAzer Technology Portfolio
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Advantages of the GeneBLAzer platform

• Robust
• Ratiometric readout reduces variation/ assay
  noise, e.g. due to cell number
• Fast
• Rapid generation of stable cell lines by flow
  cytometry
• Sensitive
• No mammalian background. Detection of lt100 BLA
  molecules / cell
• Miniaturizable
• Readily miniaturizable to 1536 3456 well
  formats

An optimal detection system for assay development
HCS
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The Cs in HCS- Cell, Content, Context
Less Searching More Content
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Why Expression Tags for In-Cell Labeling?
Live Cell Compatible Protein Specific Labeling
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Biarsenical labeling reagents bind to TC tags
Tetracysteine (TC) tag is CCPGCC (adds 1 kD)
Figure from G. Parker
TC tag 6 amino acid tag
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Tetracysteine tags are relatively small
ReAsH Tetracysteine
avGFP
595/610 nm
505/528 nm
ReAsH-EDT2
FlAsH-EDT2
Green and Red fluorescent versions
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Site-specific labeling in living cells
CFP TC-FlAsH FRET shows specificity of labeling
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Live cell imaging of mammalian cells
Low background in un-transfected neighboring cells
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Protein dynamics in living cells
Monitor cytoskeletal changes or translocation
events
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TC-FlAsH II Data - Imaging/Bkg Control
New wash protocol increase the S/N by almost 100.
Reduced Background by New Wash Protocol
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Why Tetracysteine Tag
Unique Features Application and Relevance
Easy to clone (PCR with tag in the primers) Fast project advancement (polyclonal Abs takes months)
Small tag (six amino acids) Less likely to interfere with protein function and localization
Correlation of Fluorescence microscopy with Electron microscopy High resolution detection of cellular structure
FRET- superior signals Receptor activation, protein/protein interaction
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Thank -You

• Iain Johnson
• Mike Janes
• Yih Tai Chen
• Ian Clements
• Jason Kilgore
• Dani Hill
• Jerrod Salisbury
• Kary Oakleaf
• Mike OGrady
• Alex Savtchenko
• Elena Molokanova
• Jeffrey Dzubay
• Jeff Hung
• Beth Browne
• Kathy Free
• Brett Williams

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www.invitrogen.com/iProtocol
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www.invitrogen.com/iPath
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www.invitrogen.com/Antibodies
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Literature and brochures
Easy downloads from the web, or available on
request.
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We wrote the book on fluorescence detection . . .
You can have your own copy!
www.probes.invitrogen.com
Colour your worldand see what you havent seen
before
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Questions