Bringing Science to the Surface Oris Assays: An Innovative Platform for the Study of Cell Migration & Cell Invasion AMS Biotechnology (Europe) - www.amsbio.com - info@amsbio.com UK +44 (0) 1235 828200 - CH +41 (0) 91 604 5522 - DE +49 (0) 69 77 9099
Oris and Oris Pro Assays Create a cell-free, central Detection Zone in the center of each well into which cell movement can occur. Enable the study of both cell migration (2D) and cell invasion (3D). Offer various options for pre-coated or coat-your-own Extracellular Matrix formats. Allow collection of real-time data using multiple fluorescent stains and quantify using a versatile range of image based or microplate reader instrumentation. Facilitate use of automated liquid handling equipment for cell seeding and offer unlimited access to wells from cell seeding to data readout. Pre-Migration (t= 0) Cell Migration Post-Migration (t= 21 hrs) # Cells in Detection Zone 3000 2500 2000 1500 1000 500 0 0 5 10 15 20 25 30 Time (hrs) n = 9 wells / time point
Oris Cell Motility Assay Product Line Product Name Coating Detection Zone Format Instrument Compatibility Oris Pro Cell Migration Assays Oris Pro Cell Invasion Assays Tissue Culture Treated Collagen I Coated Collagen I Coated Biocompatible Gel HCS/HCI Inverted Microscope Tissue Culture Treated Oris Cell Migration Assays Collagen I Coated Fibronectin Coated Oris Cell Seeding Stoppers (pre-populated) TriCoated Oris Cell Migration Assembly Kits Universal (TC Treated) FLEX (TC Treated) Oris Cell Seeding Stoppers (not pre-populated) HCS/HCI Inverted Microscope Microplate Reader Oris Cell Invasion Assays BME Collagen I Oris Cell Seeding Stoppers (not pre-populated) Oris Cell Seeding Stoppers (pre-populated)
Features and Benefits More Data per Well analyze cells treated with multiple fluorescent probes, labels or stains using a microplate reader, microscope or high content imaging system. Real-time Monitoring monitor changes in cell movement and morphology in real-time. Flexible Data Capture and Analysis select from a wide range of instrumentation, stains and software to achieve your experimental goals. Creative Assay Design select from our range of pre-coated plates or coat any ECM on the plate yourself -- to create a format for migration, invasion or wound closure assays. Membrane/Insert- free Cell Invasion & Migration observe cells directly without cumbersome transmembrane inserts; compatible with HTS and HCS applications. Reproducible Results achieve lower well-to-well CV s with the unique Oris assay design than with scratch assays. Z-factors attractive for HTS.
Configuring your Oris Assay Decide on a Cell Migration (2D) or Cell Invasion (3D) Assay Select the Oris Pro or Oris Format Select an ECM Choose Instrument, Method and Software for Data Capture and Analysis Optimize Assay Parameters Perform Study
Decide on a Cell Migration (2D) or Cell Invasion (3D) Assay Select the Oris Pro or Oris Format Select an ECM Choose Instrument, Method and Software for Data Capture and Analysis Optimize Assay Parameters Perform Study Decide on a Cell Migration (2D) or Cell Invasion (3D) Assay
Oris Cell Migration Assay Platform
Oris Cell Invasion Assay Platform
Select the Oris or Oris Pro Format Decide on a Cell Migration (2D) or Cell Invasion (3D) Assay Select an ECM Choose Instrument, Method and Software for Data Capture and Analysis Optimize Assay Parameters Perform Study Select the Oris Pro or Oris Format
Capabilities of Oris Pro & Oris Product Line Biocompatible Gel Oris Cell Seeding Stopper Oris Pro Cell Migration Assays Oris Cell Migration Assays Variety of ECM Coatings High Content Screening/Imaging Inverted Microscope Microplate Reader Ability to Perform: time-lapse experiments, high resolution imaging, and multiplexing using different fluorophores Compatibility with Automated Liquid Handling Equipment High Z Factor and Low Variability Compatible with Adherent Cells and Cell Migration and Invasion Formats
Oris Pro Biocompatible Gel Proven Non-Toxic Absorbance (490nm) 0.6 0.5 0.4 0.3 0.2 0.1 0.0 Oris TM Pro-Collagen I CellTiter 96 HT1080 HUVEC MDA-MB-231 No BCG BCG Fluorescence (540/590) 200 100 0 Oris TM Pro Collagen I Vibrant TM 300 No BCG BCG HT-1080 HUVEC MDA-MB-231 CellTiter 96 AQ ueous One Solution Cell Proliferation Assay (Promega) - Measures viability as metabolic function through reduction of formazan Vybrant TM Cytotoxicity Assay (Molecular Probes) - Measures glucose 6-phosphate dehydrogenase (G6PD) released from damaged cells
Equivalent Performance of Oris and Oris Pro % Closure (average) 100 75 50 25 0 Control 100 M UO126 HT-1080 cells 50 M Blebbistatin 50 M Y-27632 Oris TM Pro Collagen I Oris TM Collagen I 1 M Cytochalasin D HT-1080 cell migration after 18hr was identical in both assays (measured by % area closure of the Detection Zones). 4 different classes of cell migration inhibitors indicated substantially equivalent effects on HT-1080 cell migration in both assays.
Equivalent Performance of Oris and Oris Pro Overlapping dose-response curves and IC 50 values in Oris and Oris Pro assay formats for Cytochalasin D treated cells
Select an ECM Decide on a Cell Migration (2D) or Cell Invasion (3D) Assay Select the Oris Pro or Oris Format Choose Instrument, Method and Software for Data Capture and Analysis Optimize Assay Parameters Perform Study Select an ECM
Oris TriCoated Plate Enables Sampling of ECMs Oris Cell Migration Assay TriCoated Plate 2.8% 14.7% 3.2% Tissue Culture Treated (uncoated) Collagen I Fibronectin ECM Z' factor TC 0.79 Collagen 0.90 Fibronectin 0.31 MDS IsoCyte U0126 MEK Inhibitor
Use of Oris Assembly Kits Enables Study of ECMs
Oris Invasion Assays Permit Optimization of ECM Overlay Increasing the concentration of the BME overlay affects the ability of cells to invade.
Choose Instrument, Method and Software for Data Capture and Analysis Decide on a Cell Migration (2D) or Cell Invasion (3D) Assay Select the Oris Pro or Oris Format Select an ECM Optimize Assay Parameters Perform Study Choose Instrument, Method and Software for Data Capture and Analysis
Oris Assays Enable Versatile Data Readout Microscopes Inverted Microscopes (i.e., fluorescence, bright field, phase contrast) Fluorescence Plate Readers (bottom probe) VICTOR3 V (PerkinElmer) Synergy HT & FLx800 (BioTek Instruments) Safire2 & GENios Pro (Tecan) POLARstar Omega (BMG LABTECH) Analyst (LJL Biosystems) Paradigm (Beckman Coulter) Odyssey & Aerius Infrared Imaging Systems (LI-COR) HCS/HCI Imaging Systems Opera & Operetta (PerkinElmer) Acumen ex 3 (TTP LabTech) ArrayScan VTI HCS Reader (Cellomics) ImageXpress & Isocyte (Molecular Devices) Pathway Bioimager (BD Biosciences) IN Cell Analyzer (GE)
Area Closure to Examine Cell Migration in Detection Zone PE Operetta
Cell Count to Assess Cell Migration in Detection Zone PE Opera
Microplate Cytometer to Analyze Cell Migration in Detection Zone HT-1080 Cell Migration (µm 2 ) TTP LabTech Acumen ex 3 27
Flexible Stain Options to Measure Cell Migration DAPI Calcein AM TRITC-phalloidin Control 16 hour serum Fluorescent Label Z S:N DAPI 0.48 19.03 Calcein AM 0.50 22.79 TRITC-phalloidin 0.50 18.73 Z-factor of J Biomol Screen. - Oris Assays achieve similar Z factors on microplate cytometers using DAPI, Calcein AM and TRITC-phalloidin stains
Convenient Assay Options to Measure Endpoint Cell Migration Cell Migration Images: MDA-MB-231 4000 Cell Migration Data: MDA-MB-231 Oris Cell Migration Assay Collagen I Coated Kit Mean Migration (RFUs) 3500 3000 2500 2000 1500 1000 500 0 0 hr 20 hr Average RFU 985 3165 S.D. 61 238 % CoV 6.2 7.5 Signal-Noise 8.9 Z-factor 0.59
Dynamic Assay Options to Measure Kinetic Cell Migration 3 sirna pools against different PtdIns pathway targets. Fluorescence Readings obtained for 48 hr (every 20 min) for cells migrating on fibronectin. Data presented as average AUC. Cells transfected with sirna pools A & B demonstrated a similar drop in migration. BMG LABTECH POLARstar Omega sirna transfected U2OS cells labeled with DiI-C16 sirna pool C had a more pronounced knockdown effect than pools A & B.
ImageJ Software Compatibility to Determine Cell Number and Area Closure Particle Analysis - DAPI Stain (MDA-MB-231 Cells) Area Closure Analysis - Phase Contrast (HT-1080 Cells)
Optimize Assay Parameters Decide on a Cell Migration (2D) or Cell Invasion (3D) Assay Select the Oris Pro or Oris Format Select an ECM Choose Instrument, Method and Software for Data Capture and Analysis Perform Study Optimize Assay Parameters
Oris Assay Optimization Parameters Identify cell seeding density to achieve 95% -100% confluence upon initiation of migration. Choose appropriate ECM for your cells. Determine the appropriate time needed to observe robust cellular movement into Detection Zone. Accurately acquire data by selecting appropriate stain (live vs. fixed; nuclear vs. cytoskeletal) and choice of instrumentation. F-actin and Focal Adhesion Staining
Identify Appropriate Cell Seeding Density Acceptable Not Acceptable A549 Cells Cellomics ArrayScan
Perform Study Decide on a Cell Migration (2D) or Cell Invasion (3D) Assay Select the Oris Pro or Oris Format Select an ECM Choose Instrument, Method and Software for Data Capture and Analysis Optimize Assay Parameters Perform Study
Oris Assays Enable Cell Migration Analysis of c-met Pathway Migration of Calcein AM stained A549 cells on Collagen I coated plates was quantitated by use of a BioTek Synergy fluorescence microplate reader. [PHA-665752] (Log M)
Multiple Approaches to Studying c-met Inhibition using Cell Migration
Multiplexed Assay of ERK Phosphorylation and its Impact on Cell Migration Cell Migration (RED) ERK Phosphorylation (GREEN) Simultaneous determination of effects of MEK and PI 3 kinase inhibition on cell migration (IRDye680 RED) and ERK phosphorylation (IRDye800cw GREEN) using multiplexed staining in a LI-COR Odyssey system with the Oris Detection Mask attached. Cell Migration is inhibited equivalently by the MEK inhibitor U0126 and the PI 3 kinase inhibitor Ly294002 while inhibition of ERK phosphorylation is more effectively and completely blocked by the MEK inhibitor U0126 than by the PI 3 kinase inhibitor Ly294002.
Oris Cell Invasion Assays Provide a 3-D Environment Invasive - T=0 hrs Non-Invasive -Swiss Albino Cells T=48 hrs (no FBS) T=48 hrs (10% FBS) T=0 hrs T=48 hrs (no FBS) T=48 hrs (10% FBS) Mean Fluorescence Units 2000 1600 1200 800 400 0 0hrs 48hrs no FBS 10% FBS no FBS 10% FBS ---------HT1080 cells--------- ---------3T3 cells------- Supplements in the Oris BME overlay
Oris Invasion Assays Enable Characterization of Invadopodia A. B. C. Indirect Immunofluorescence to Identify Invadopodia Markers: F-actin and Cortactin co-localized (A) Expression of MMP-9 protease (B) Expression of Cathepsin B (C) HT-1080 cells were imaged after 48 hours of 3-D invasion into BME.
Oris Invasion Assays Capture Cells in the Z-axis HT-1080 Cell Invasion in Oris Pro Collagen Invasion Assay
Oris Invasion Assays Capture Cells in the Z-axis HT-1080 Cells 72 h Invasion 4 mg/ml Collagen I Number of Invaded Cells Microns Above Plate Surface
Oris Invasion Assays Help Unravel Complex Signal Transduction Mechanisms TGF- 1 & EGF synergize to promote HaCaT II-4 invasion into BME EGFR Inhibitor P38 Kinase Inhibitor MEK Inhibitor Signal transduction inhibitors attenuate TGF- 1 & EGF induced invasion Plasminogen Activator Inhibitor -1 (PAI-1) sirna knockdown attenuates HaCaT II-4 invasion into BME in both basal and stimulated conditions
Cell Motility Assay Competition - Scratch Assays Oris Assay Area Closure: 87-89% Scratch Assay Area Closure: 69-77% Oris Assay %CV: 3.7-6.5% Scratch Assay % CV: 11.3-25.6% Oris Assay: ECM intact Scratch Assay: scratching damages the ECM
Cell Motility Assay Competition Transmembrane Assays Oris Assays permit real-time visualization and multiplexed staining of cells. Oris Invasion Assays provide a more physiologically relevant 3D environment and allow collection of Z-stack images. Oris Assays allow rapid evaluation using a microplate reader.
Cell Motility Assay Comparison Scratch Assays Transmembrane Assays Oris Assays Benefits Limitations Cells are Damaged During Scratch Formation ECM can be Damaged When Creating Scratch Inconsistent Scratch Size Cannot Use Microplate Reader Cells Move in Defined Direction Choice of ECM High Resolution Images are Difficult to Obtain Cannot Perform Time-Lapse Experiments Variable Results Provide Chemical Gradient Choice of ECM Ability to Use Microplate Reader Not Compatible with Non-Adherent Cells Not Suitable for Chemotaxis High Resolution Imaging Multiplex Using Different Fluorophores Flexibility in Readout (Microplate Reader, Inverted Microscope, High Content Screening & High Content Imaging) Choice of ECM
New - Oris Pro 384 Miniaturization of Oris Pro to 384-well assay Particularly well-suited for higher density screening of compound libraries Seed BCG Dissolves to Reveal Detection Zone Analyze Cell Migration by HCS/HCI Instrumentation Achieve Robust Z 10,000 cells/well; 2.5 x magnification; TRITC-phalloidin stained HT-1080 cells Z = 0.76 after 19 h migration on TC treated surface
How Oris Assays Can Work for You Assay formats are suitable for partial or full automation. Offer flexibility to select the most optimal combination of instrumentation, stains and software for data capture and analysis. Enables the choice of migration (2D) and invasion (3D) assay formats. Select the ECM best suited for your experiment. Application and Technical Support fromamsbio and instrument vendors. Demonstrated capabilities for both target analysis and compound screening.
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