Bringing Science to the Surface™ Oris™ Assays: An ... Cell Migration and Invasion Assays... ·...
Transcript of Bringing Science to the Surface™ Oris™ Assays: An ... Cell Migration and Invasion Assays... ·...
Bringing Science to the Surface™
Oris™ Assays: An Innovative Platform
for the Study of Cell Migration & Cell Invasion
AMS Biotechnology (Europe) - www.amsbio.com - [email protected]
UK +44 (0) 1235 828200 - CH +41 (0) 91 604 5522 - DE +49 (0) 69 77 9099
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.
0
500
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0 5 10 15 20 25 30Time (hrs)
n = 9 wells / time point#
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lls
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ePre-Migration
(t= 0)
Post-Migration
(t= 21 hrs)
Cell Migration
Oris™ and Oris™ Pro Assays
Oris™ Cell Motility Assay Product Line
Product Name CoatingDetection Zone
Format
Instrument
Compatibility
Oris™ Pro
Cell Migration Assays
Tissue Culture Treated
Collagen I CoatedBiocompatible Gel
HCS/HCI
Inverted MicroscopeOris™ Pro
Cell Invasion Assays
Collagen I Coated
Oris™ Cell Migration
Assays
Tissue Culture Treated
Collagen I Coated
Fibronectin Coated
TriCoated
Oris™ Cell Seeding Stoppers
(pre-populated)
HCS/HCI
Inverted Microscope
Microplate Reader
Oris™ Cell Migration
Assembly Kits
Universal (TC Treated)
FLEX (TC Treated)
Oris™ Cell Seeding Stoppers
(not pre-populated)
Oris™ Cell Invasion
Assays
BMEOris™ Cell Seeding Stoppers
(not pre-populated)
Collagen I Oris™ Cell Seeding Stoppers
(pre-populated)
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.
Features and Benefits
Configuring your Oris™ Assay
Select the Oris™ Pro or
Oris™ 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
ParametersPerform Study
Select an ECM
Choose
Instrument,
Method and
Software for
Data Capture
and Analysis
Optimize Assay
ParametersPerform Study
Decide on a Cell Migration (2D) or Cell Invasion (3D) Assay
Select the Oris™ Pro or
Oris™ 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
ParametersPerform Study
Select the Oris™ or Oris™ Pro Format
Select the Oris™ Pro or
Oris™ Format
Decide on a
Cell Migration
(2D) or Cell
Invasion (3D)
Assay
Capabilities of Oris™ Pro & Oris™
Product Line
Oris™ Pro
Cell Migration Assays
Oris™
Cell Migration Assays
Biocompatible Gel
Oris™ Cell Seeding Stopper
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
OrisTM Pro-Collagen I
CellTiter 96
HT1080 HUVEC MDA-MB-2310.0
0.1
0.2
0.3
0.4
0.5
0.6No BCG
BCG
Ab
so
rban
ce (
490n
m)
OrisTM Pro Collagen I
VibrantTM
HT-1080 HUVEC MDA-MB-2310
100
200
300 No BCG
BCG
Flu
ore
scen
ce (
540/5
90)
CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega)- Measures viability as metabolic function through reduction of formazan
VybrantTM Cytotoxicity Assay (Molecular Probes)- Measures glucose 6-phosphate dehydrogenase (G6PD) released from
damaged cells
HT-1080 cells
Contr
olM
UO
126
100
M B
lebbis
tatin
50
M Y
-276
32
50
M C
ytoch
alas
in D
1
0
25
50
75
100Oris
TM Pro Collagen I
OrisTM
Collagen I
% C
losu
re (
avera
ge)
Equivalent Performance of Oris™ and Oris™ Pro
• 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.
Overlapping dose-response curves and IC50
values in Oris™ and Oris™ Pro assay formats
for Cytochalasin D treated cells
Equivalent Performance of Oris™ and Oris™ Pro
Select an ECM
Choose
Instrument,
Method and
Software for
Data Capture
and Analysis
Optimize Assay
ParametersPerform Study
Select an ECM
Select the Oris™ Pro or
Oris™ Format
Decide on a
Cell Migration
(2D) or Cell
Invasion (3D)
Assay
Oris™ TriCoated Plate Enables Sampling of ECMs
2.8% 14.7% 3.2%
MDS IsoCyte™
Tissue Culture
Treated (uncoated)
Collagen I
Fibronectin
Oris™ Cell Migration Assay TriCoated Plate
ECM Z' factor
TC 0.79
Collagen 0.90
Fibronectin 0.31
U0126 MEK Inhibitor
Oris™ Invasion Assays
Permit Optimization of ECM Overlay
Increasing the concentration of the BME overlay
affects the ability of cells to invade.
Select an ECM
Choose
Instrument,
Method and
Software for
Data Capture
and Analysis
Optimize Assay
ParametersPerform Study
Choose Instrument, Method and Software for Data Capture and Analysis
Select the Oris™ Pro or
Oris™ Format
Decide on a
Cell Migration
(2D) or Cell
Invasion (3D)
Assay
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® eX3 (TTP LabTech)
• ArrayScan® VTI HCS Reader (Cellomics)
• ImageXpress™ & Isocyte™ (Molecular Devices)
• Pathway™ Bioimager (BD Biosciences)
• IN Cell Analyzer (GE)
Oris™ Assays Enable Versatile Data Readout
27
Microplate Cytometer to Analyze
Cell Migration in Detection Zone
TTP LabTech Acumen eX3
HT-1080
Cell Migration
(µm
2)
Control
16 hour
serum
DAPI Calcein AM TRITC-phalloidin
Fluorescent Label Z’ S:N
DAPI 0.48 19.03
Calcein AM 0.50 22.79
TRITC-phalloidin 0.50 18.73
Flexible Stain Options
to Measure Cell Migration
Oris™ Assays achieve similar Z’ factors on microplate cytometers
using DAPI, Calcein AM and TRITC-phalloidin stains
Z-factor of
J Biomol Screen. -
Convenient Assay Options
to Measure Endpoint Cell Migration
Average RFU 985 3165
S.D. 61 238
% CoV 6.2 7.5
Signal-Noise 8.9
Z-factor 0.59
0
500
1000
1500
2000
2500
3000
3500
4000
0 hr 20 hr
Mean
Mig
rati
on
(R
FU
s)
Cell Migration Data: MDA-MB-231
Oris™ Cell Migration Assay
Collagen I Coated Kit
Cell Migration Images: MDA-MB-231
• 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.
• siRNA pool C had a more
pronounced knockdown
effect than pools A & B.
Dynamic Assay Options
to Measure Kinetic Cell Migration
BMG LABTECH POLARstar Omega siRNA transfected U2OS
cells labeled with DiI-C16
Particle Analysis - DAPI Stain (MDA-MB-231 Cells) Area Closure Analysis -
Phase Contrast (HT-1080 Cells)
ImageJ Software Compatibility
to Determine Cell Number and Area Closure
Select an ECM
Choose
Instrument,
Method and
Software for
Data Capture
and Analysis
Optimize Assay
Parameters
Perform Study
Optimize Assay Parameters
Select the Oris™ Pro or
Oris™ Format
Decide on a
Cell Migration
(2D) or Cell
Invasion (3D)
Assay
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
Select an ECM
Choose
Instrument,
Method and
Software for
Data Capture
and Analysis
Optimize Assay
Parameters
Perform Study
Perform Study
Select the Oris™ Pro or
Oris™ Format
Decide on a
Cell Migration
(2D) or Cell
Invasion (3D)
Assay
Oris™ Assays Enable Cell Migration
Analysis of c-Met Pathway
[PHA-665752] (Log M)
Migration of Calcein AM stained A549 cells
on Collagen I coated plates was quantitated
by use of a BioTek Synergy™ fluorescence
microplate reader.
• Simultaneous determination of effects of MEK and
PI3 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 PI3 kinase inhibitor Ly294002
while inhibition of ERK phosphorylation is more
effectively and completely blocked by the MEK
inhibitor U0126 than by the PI3 kinase inhibitor
Ly294002.
Multiplexed Assay of ERK Phosphorylation
and its Impact on Cell Migration
Cell Migration (RED) ERK Phosphorylation (GREEN)
T=0 hrs T=48 hrs (no FBS) T=48 hrs (10% FBS)
Non-Invasive -Swiss Albino Cells
Oris™ Cell Invasion Assays
Provide a 3-D EnvironmentInvasive -
T=48 hrs (no FBS) T=48 hrs (10% FBS)T=0 hrs
0
400
800
1200
1600
2000
no FBS 10% FBS no FBS 10% FBS
Supplements in the Oris™ BME overlay
Me
an
Flu
ore
sce
nce
Un
its
0hrs
48hrs
---------HT1080 cells--------- ---------3T3 cells-------
Oris™ Invasion Assays
Enable Characterization of Invadopodia
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.
C.A. B.A. B.
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
Microns Above Plate Surface
Nu
mb
er
of
Inva
de
d C
ell
s
• HT-1080 Cells
• 72 h Invasion
• 4 mg/mL Collagen I
Oris™ Invasion Assays Help Unravel
Complex Signal Transduction Mechanisms
EGFR Inhibitor P38 Kinase Inhibitor MEK Inhibitor
• TGF- 1 & EGF synergize
to promote HaCaT II-4
invasion into BME
• 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
• 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 Competition –
Transmembrane Assays
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
Be
ne
fits
L
imit
ati
on
s
Scratch Assays Transmembrane Assays Oris™ Assays
Cell Motility Assay Comparison
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 from amsbio and instrument
vendors.
• Demonstrated capabilities for both target analysis and compound
screening.