Why Doing Live-Cell Imaging?

Transcription

1 Why Doing Live-Cell Imaging?

2 The Snapshot Bias Different Endpoint = Different Result

3 IncuCyte System Key Advantages

4 IncuCyte Live-Cell Imaging System

5 Software Advantage: Distributed Access NETWORK Unlimited licenses Remote control & access Database management Integrated analysis

6 1000s of Users, 100s of Peer Reviewed Publications and Growing Cancer Biology Immunology Neuroscience Virology Stem Cell Biology 3D Biology

7 IncuCyte ZOOM Key Applications Confluence, QC & Cell Health Cell Migration & Invasion Proliferation & Cell Counting Chemotaxis Migration & Invasion Adherent Chemotaxis Migration Suspension 3D-Spheroids Apoptosis Cytotoxicity T-cell killing Phagocytosis Gene Expression Neurite Dynamics Clonal Dilution end Angiogenesis Stem Cell Monitoring & Reprogramming

8 Cell Confluence & Health HT1080 fibrosarcoma cells Measure cell proliferation over time & without labels Cells remain in your incubator throughout assay Applicable to a wide range of cell types Validate data and morphology with images & movies Throughput Time course data Microplate view

9 Cell Migration & Invasion WoundMaker HT1080 cells, Matrigel Biomatrix ImageLock 96-well Plates IncuCyte Cell Migration Software Module Create scratch wounds in seconds on 96-well plates Automatically analyse full time course of wound healing No labels required, amenable to wide range of cells Multiplex migration and invasion through ECM Throughput Time course data Microplate view

10 Cell Proliferation and Cell Counting Single EF-1 alpha promoter used to express both fluorescent label and puromycin resistance marker NucLight Red HT1080 Day 1: Plate cells in flasks or plates Day 2: Add NucLight Reagent Day 3: Replace media Day 4: Proliferation Assay as transient expressing culture, if primary cells Days 5-12: Apply drug selection (puromycin) to derive a stable, homogeneous population New Stable Cell Line for use in Proliferation Assay Create new cell lines or transient labelled primary cultures with red or green nuclei Direct, linear quantitation and that is not subject to artefacts Can be multiplexed with Apoptosis or other cell health assays Cell Lines also available in Green or Red from Essen NucLight Red HT1080 and NucLight Green A549 grown in co-culture NucLight Green Count/mm Time (h) Throughput NucLight Green A549 Untreated 4.12nM 12.35nM 37.04nM nM nM 1000nM 3000nM

11 Chemotaxis & Directional Migration CellPlayer 96-well ClearView Cell Migration Plate (Cat No 4582) HT 1080 Area Normalized to Initial Top Value Time (h) Time course data Throughput HT 1080 fibrosarcoma cells Visualize cells migrating toward chemo-attractants No labels required, low cell usage, surface interactions Monitor full time course of chemotaxis Set it & forget it fully automated analysis Microplate view 10

12 Chemotaxis & Directional Migration Jurkat T lymphocyte cells IncuCyte ClearView Cell Migration Plate Visualize cells migrating toward chemo-attractants No labels required, low cell usage, surface interactions Monitor full time course of chemotaxis Set it & forget it fully automated analysis Throughput C5a fmlp Time course data IL-8 Microplate view 11

13 3-D Spheroids t=0 t=96h t=198h t=258h Vehicle Control SPP 1 mm OX 100 mm A549 Spheroids CHX 10 mm Ultra Low Attachment Round Bottom Plates used Time course data Dose response to Staurosporine Throughput Staurosporine Oxaliplatin Cycloheximide Media/DMSO Spheroids creation easy Area and fluorescence intensity metrics used to measure growth, shrinkage and spheroid health Fluorescence Area (µm 2 ) Validate findings with images and movies Time (days) Microplate view

14 Apoptosis IncuCyte Caspase-3/7 Apoptosis Assay Reagent HT1080 cells + Camptothecin + Caspase-3/7 reagent Throughput Time course data Quantify real time commitment to death by apoptosis No wash, mix and read assays in 96- and 384-well format Easily duplex with cell proliferation measures Validate data with images & movies Microplate view

15 Cytotoxicity Kinetic Cytotoxicity using IncuCyte Cytotox Red and IncuCyte Cytotox Green Reagents X cells + Y Drug+ YOYO-1 Throughput Time course data Quantify real time cell death No wash, mix and read assays in 96- and 384-well format Easily duplex with NucLight Red Proliferation Assay Validate data with images and movies Microplate view

16 Immune Cell Killing (T cell, ADCC) NucLight Red SK-OV-3 cells + PBMCs + Caspase 3/7 reagent Tumor cell apoptosis (green) Tumor cell count (red) IncuCyte Caspase-3/7 Apoptosis Assay Reagent NucLight Red Throughput Time course data Direct, kinetic measures of tumor cell death Mix-and-read protocols - no radioactivity or cell lifting Images/movies validate target/effector cell interactions T cell killing, ADCC - your choice of effector/target cells Microplate view

17 Immune Cell Clustering & Proliferation Immune cell Clustering T cell clustering 0 h 72 h 144 h PBMCs activated with anti-cd3 antibody and IL-2 Immune cell Proliferation T cell proliferation 0 h 72 h 144 h PBMCs activated with anti-cd3 antibody and IL-2 Monitor proliferation and clustering in real-time Automated imaging and time-course analysis Label free, kinetic measurements No wash, no cell lifting, no antibody labelling Phase object count (per mm 2 ) mg/ml 2.5 mg/ml 0.63 mg/ml 0.01 mg/ml IL-2 (10 ng/ml) Time (h) Time course data

18 Phagocytosis & IncuCyte phrodo Bioparticles Fluo Fluo +Phase J774A.1 macrophages + IncuCyte phrodo Green E. coli bioparticles Green Object Area (µm² x10 5 /mm 2 ) Time (hours) Throughput 10K cells/well 3K cells/well 1K cells/well 0 cells/well Cell number dependence Validation of phagocytosis using time-lapse imaging Automated analysis and quantification Mix-and-read; no fixing, no quenching, no lifting Highly sensitive, low background, low cell numbers 18

19 Reporter Gene Example: TNFα Activation of NF-κB Ligand (TNFα) TNFα Receptor GFP Cells transfected with a reporter gene for NF-κB treated with TNFα Signaling pathway affected - active NF-κB produced Active NF-κB p50 RelA NF-κB binding sequence in promoter region Reporter Gene Dose response of TNFα added to cells transfected with NFκB-GFP reporter genes is shown in this example. The activation of the TNF receptor leads to the activation of NF-κB, a transcription factor that, in turn, stimulates gene expression. After the addition of the TNFα, the cells are placed into the IncuCyte ZOOM which kinetically measures GFP expression. Time course dose response to TNFα addition Research Area Cancer Inflammation Cardiovascular Research Stem Cells Nuclear Receptors Toxicity Reporter Constructs (Transcription Factor) NF-κB, p53, MAPKs, Wnt, HIF-1α, PI3K NF-κB, TGF-β, Interferon Response camp/pka, Notch, MEF-2 KLF4, Oct4, Nanog PPARγ AhR (CYPs), Nrf1, Nrf2

20 Neurite Dynamics Neurite outgrowth Throughput Time course data Monitor neurite outgrowth over days and weeks Label-free automated analysis in mono-culture Specialized neuronal fluorescent labels for co-culture Validated for primary & ipsc-derived neurons Microplate view

21 Clonal Dilution Monitoring Software Demo Step 1: Image plates inside IncuCyte for extended periods. Automatic colony identification without cell labels. Step 2: Inspect colony morphology Step 3: Confirm clonality by browsing backward and forward in time Marking Tool Automated image processing to Identify and track clones, confirm clonality Mark clones in software, physically mark plates with IncuCyte Marking tool Automated image panning of large vessels such as 35mm dishes Throughput Six 96 or 384 Microplates can be imaged in parallel

22 Angiogenesis ADSC/ECFC Cord Formation Essen BioScience Stem Kit for Angiogenesis NHDF/HUVEC Tube Formation Two well published Angiogenesis co-culture models consisting of cryopreserved cells and supplements available from Essen BioScience: 1) Adipose Derived Stem Cells/Endothelial Colony Forming Cells (model yields rapidly forming (<48h) endothelial cell cord structures) 2) Normal Human Dermal Fibroblasts/HUVEC (slowly forming tubelike structures appear, grow and branch for 10 days) 3) Different morphological, temporal, and pharmacological profiles

23 Stem Cell Reprogramming Day 0 Add Sendai virus containing Yamanaka Factors Day 13 Colonies begin to emerge Days Transfer ipsc colonies Day -2 Plate cells (Neonatal or Adult fibroblasts) Day 7 Passage cells (MEFs or Feeder Free) Cell inspection & refeed Stem cell colony 12 days post passage Colony Marking and Tracking Days Evaluate toxicity of reprogramming reagents Days Track colony emergence, growth, and morphology - Standardize calculation of reprogramming efficiencies IncuCyte Marking Tool Days Post-Reprogramming - monitor clone transfer and survival

24 IncuCyte ZOOM Key Features Multiple Imaging Modes HD phase-contrast, green & red fluorescence, 4x 10x 20x Sample area to whole well image capture, up to 6 plates at once 24h scheduling and scan on demand Multi-Vessel, Multi-User Compatible with over 600 standard plates, dishes & flasks Mix & match vessels & assays (slides, dishes, flasks, and 6 to 384-well plates) Remote Viewing & Analysis Fully automated, fast on the fly analysis Shared access via any networked Windows device

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