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

1000

1500

2000

2500

3000

0 5 10 15 20 25 30Time (hrs)

n = 9 wells / time point#

Ce

lls

in

De

tec

tio

n Z

on

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


Decide on a Cell Migration (2D) or Cell Invasion (3D) Assay


Oris™ Format

Decide on a

Cell Migration

(2D) or Cell

Invasion (3D)

Assay

Oris™ Cell Migration Assay Platform

Oris™ Cell Invasion Assay Platform

Select an ECM

Choose

Instrument,

Method and

Software for

Data Capture

and Analysis

Optimize Assay


Select the Oris™ or Oris™ Pro Format


Oris™ Format

Decide on a

Cell Migration

(2D) or Cell

Invasion (3D)

Assay

Capabilities of Oris™ Pro & Oris™

Product Line

Oris™ Pro


Oris™


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


Select an ECM


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

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.

Select an ECM

Choose

Instrument,

Method and

Software for

Data Capture

and Analysis

Optimize Assay


Choose Instrument, Method and Software for Data Capture and Analysis


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

Area Closure to Examine Cell Migration in

Detection Zone

PE Operetta™

Cell Count to Assess

Cell Migration in Detection Zone

PE Opera™

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


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

Identify Appropriate Cell Seeding Density

Cellomics ArrayScan®

A549 Cells

Acceptable Not Acceptable

Select an ECM

Choose

Instrument,

Method and

Software for

Data Capture

and Analysis

Optimize Assay

Parameters

Perform Study

Perform Study


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.

Multiple Approaches to Studying

c-Met Inhibition using Cell Migration

• 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.

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bringing science to the surface™ oris™ assays: an ... cell migration and invasion assays... ·...

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