Mechanisms of Cell MigrationAntibodies, Proteins, Kits and Assays

Data SheetProduct Selection Guide

THE EXPERTISE OF UPSTATE®, CHEMICON® & LINCO®

IS NOW A PART OF MILLIPORE

As a tools provider and partner in research, Millipore is committed to the advance-

ment of life science research and therapeutic development. This guide includes a

number of new products for target identification, pathway detection and profiling.

These products provide proven solutions for a range of applications and are backed

by expert technical support.

Platforms and Technologies

Antibodies and ImmunoassaysMillipore offers an extensive, focused portfolio of antibodies and immunoassays. With

the expertise of Upstate® and Chemicon®, Millipore provides validated products with

breadth and depth in major research areas backed by excellent service and support.

Cell Based Assays and High Content AnalysisMillipore offers a significant portfolio of live cell, whole-cell and cell-based activity assays

and reporter systems for direct and indirect detection. These technologies facilitate

protein target validation, identify cellular pathways and determine mechanism of action

for lead optimization environments. Millipore also offers an array of assays for high-

content multi-parametric analysis; enabling identification of cellular responses and

events under user-defined conditions.

Flow Cytometry Assays and SystemsFlow cytometry is an essential tool for in-depth cell analysis, with the capacity to

simultaneously measure multiple parameters on individual cells. Guava® flow cytometers

provide direct, precise measurement via microcapillary technology that translates into

smaller samples, less reagents, and minimal waste. Millipore also offers FlowCellect™

reagents and kits that are optimized for guava systems and compatible with traditional

core lab environments, along with application-specific analysis software modules, to

provide a complete solution for flow cytometry.

MILLIPLEX® MAP Multiplex AssaysMILLIPLEX MAP assays offer the broadest selection of multiplex kits and reagents in a

wide variety of therapeutic areas, measuring multiple biomarkers using a small sample

size. Compared to conventional methods, such as ELISAs and Western blots, MILLIPLEX MAP

enables the simultaneous detection of multiple soluble or intracellular biomarkers. Using

the Luminex® xMAP® bead-based technology, Millipore’s flexible and customizable assays

are exhaustively tested and qualified for sensitivity, specificity, reproducibility and wide

dynamic range.

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IntroductionCell Movement Recent studies elucidating the effect of extracellular environment on cell

movement have revolutionized studies of diverse processes, including stem

cell differentiation, morphogenesis, wound healing, cancer metastasis, and

immune response. In just the last few years, discoveries have shown that cell

movement can be characterized by multiple, interchangeable parameters

that can be adjusted dynamically by factors such as cell type, multipotency,

and adhesive forces. As a result, the study of cell movement now depends on

a powerful confluence of research areas and experimental techniques.

Millipore’s life scientists possess a unique breadth and depth of research

focus, as well as expertise in a vast array of cell analysis methods, enabling

us to offer the only complete solution for studying cell structure,

environment, and movement.

Cell motility is an integrated process that is carefully and precisely

orchestrated by the cell with the help of many receptors, cross linking,

bundling, binding, adhesion, motor, and other proteins. These proteins

ultimately determine the direction of cell movement, carrying out the

motility steps in an exactly timed manner. The key steps in cell movement

are as follows: (1) interaction with the environment (2) signaling, (3)

cytoskeletal rearrangement, and (4) rearrangement of adhesive contacts to

the substratum. External signals detected by receptor proteins located on

the cell membrane, which transmit the signals to the cytoskeleton and

nucleus via intracellular signaling pathways. The cytoskeleton is a complex,

dynamic network consisting of three types of polymers: actin, microtubules,

and intermediate filaments. In response to the signal, the cell rearranges

both the cytoskeleton as well as the set of adhesive contacts between the

cell and the environment. This enables the cell to start migrating.

A thorough understanding of the mechanisms regulating cell movement

is essential for gaining deeper insight into morphogenesis, wound healing,

neurogenesis, immune response, and other processes. In addition, cell

movement is central to tumor progression and metastasis, in which cells

from a primary tumor move away and spread to other parts of the body.

Understanding how the cell generates the forces necessary for movement

and learning how these forces operate at the microscopic scale will

ultimately facilitate incorporating similar principles in engineering

microscopic-scale tools for both research and therapeutics.

With this future potential in mind, Millipore is continuously expanding its

comprehensive portfolio of cell structure research tools to keep our

customers at the forefront of the field. This product guide represents a

sample of some of our most exciting new offerings for studying cell

movement.

Table of Contents

MIGrATIon

Chemotaxis 5• QCM™ Chemotaxis Migration

Assay (fluorometric)• QCM Endothelial Cell Migration

Assay (fluorometric)

Invasion 6• QCM Laminin Migration Assay

(fluorometric) • Millicell® and MultiScreen® -

MIC Systems

Featured Product:Millicell® µ-Angiogenesis Kits

CyToSKELETAL SIGnALInG

Signaling Proteins 9• Phospho-FAK (Tyr397)

STAR ELISA Kit • Ras GTPase Activation

ELISA Kit

Cytoskeleton 10• Actin Cytoskeleton and

Focal Adhesion Staining Kit• Acetyl-Cortactin Antibody

Featured Product:AXIS™ Axon Investigation System

ADHESIon

Cell to ECM Interactions 13• a Integrin-Mediated Cell

Adhesion Colorimetric Array Kit

Cell to Cell Interactions 14• Endothelial Cell Adhesion Assay• ECM Cell Adhesion Array

Featured Product:QCM Leukocyte Transendothelial Cell Migration Assay

EXTrACELLULAr MATrIX & ASSoCIATED ProTEASES

ECM 17• ECM Cell Culture

Optimization Array• ECM Proteins and Antibodies

Proteases MMPs/TIMPs 18• MT1-MMP Antibody• GM6001 MMP Inhibitor

Featured Product:MILLIPLEX® MAP Human Bone Multiplex Panel

4

Cell migration is stimulated and directed by interaction of cells with

the extracellular matrix (ECM), neighboring cells, or chemoattractants.

During embryogenesis, cell migration participates in nearly all

morphogenic processes ranging from gastrulation to neural

development. In the adult organism, cell migration contributes to

physiological and pathological conditions, and is central to

development of therapeutics affecting wound healing and tumor

metastasis. Specifically, inhibiting tumor invasion by blocking tumor

cell chemotaxis has been a major focus of research.

Migration

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QCM™ Chemotaxis Cell Migration Assay (96-well, fluorometric) (Catalogue No. ECM510)

The chemotaxis cell migration assay measures directional cell

movement in response to chemical concentration gradients.

While multiple pore sizes are available, the 8 µm pore size of

this assay supports optimal migration for most epithelial and

fibroblast cells.

Chemotaxis

Human fibrosarcoma HT-1080 chemotaxis toward 10% fetal bovine serum

(FBS) was measured in the presence or absence of cytochalasin D, an

inhibitor of actin polymerization. Note that cytochalasin D inhibits cell

migration towards chemoattractant FBS. Assayed at multiple time intervals.

Migration assay using HUVECs plated on chambers coated with bovine

serum albumin (BSA) or fibronectin (FN). Fetal bovine serum functions as an

effective chemoattractant, stimulating cell migration on FN but not on BSA.

Chemotaxis assays are ideal for assessing the effects of pharmacological compounds on the motility

of tumor cells, and for analyzing the migratory capacity of multiple cell lines in parallel. The most

widely accepted cell migration assay is the Boyden chamber assay. Millipore’s system uses a

two-chamber multiwell plate in which a membrane in each well provides a porous interface between

two chambers. This user-friendly, high throughput format enables sensitive, quantitative comparison

of multiple samples.

QCM Endothelial Cell Migration Assay (24-well, fluorometric) (Catalogue No. ECM201)

Angiogenesis, the formation of blood vessels, results from

migration of endothelial cells and is regulated by ECM

components, angiogenic, and anti-angiogenic factors. The

endothelial cell migration assay provides a quick and efficient

system to study a compound’s ability to induce or inhibit

endothelial cell migration.

KEy ProDUCTS

Description Catalogue No.

QCM Endothelial Cell Migration Assay ECM200

(24-well, colorimetric)

QCM Endothelial Cell Migration Assay ECM202

(96-well, fluorometric)

QCM Chemotaxis Assay (24-well, 3 µm, colorimetric) ECM504

QCM Chemotaxis Assay (24-well, 3 µm, fluorometric) ECM505

QCM Chemotaxis Assay (24-well, 5 µm, colorimetric) ECM506

Description Catalogue No.

QCM Chemotaxis Assay (24-well, 5 µm, fluorometric) ECM507

QCM Chemotaxis Assay (24-well, 8 µm, colorimetric) ECM508

QCM Chemotaxis Assay (24-well, 8 µm, fluorometric) ECM509

QCM Chemotaxis Assay (96-well, 5 µm, fluorometric) ECM512

QCM Chemotaxis Assay (96-well, 3µm, Fluorometric) ECM515

Fibroblast Growth Factor basic, human recombinant GF003

(multiple)

HT-1080 Chemotaxis Assay in 96-well Format

0

50

100

150

200

250

RFU

x 1

00

0

300

2h 4h 24h

0 % FBS

10 % FBS

10 % FBS + 10 µM Cytochalasin D

0

25

50

RFU

x 10

00

75

100

125

150

175

BSA BSA+FBS FN+FBSFN

HUVEC Migration on Fibronectin Coated Inserts

6

QCM Laminin Migration Assay (24-well, fluorometric) (Catalogue No. ECM221)

The ECM protein laminin, promotes cell adhesion,

proliferation, and migration of several cell types via

interaction with integrin heterodimers a1b1, a2b2, a3b1,

a6b1, a7b1, and a6b4. This assay is a simple, effective

screening tool to quantify the migration of neural cells,

embryonic stem cells, and various cancer cells.

InvasionThe basement membrane surrounding the blood vessel endothelium is a thin, specialized network of

ECM proteins. The ability of tumor cells to degrade the ECM components of the basement membrane

and surrounding tissues is directly correlated with metastatic potential. QCM cell invasion assays

enable convenient and sensitive quantification of in vitro cell invasion through a basement

membrane model. A Boyden chamber system is layered with an ECM solution that occludes the

membrane pores, blocking non-invasive cells from migrating through it.

ReNcell™ CX human cortical neural stem cells (SCC007) and breast cancer cell

line, MDA-MB-231, demonstrate laminin-mediated migration (dark green bars).

Note that BSA does not induce migratory response from either cell type.

Millicell® Inserts and MultiScreen®-MIC System The Millicell inserts and MultiScreen-MIC filter plates provide reliable, versatile devices for a range of cell-based screening assays

including migration, invasion, chemotaxis, co-culture, angiogenesis, and transmigration. Inserts and plates are available in a range of

pore sizes to support assays with suspension or adherent cell lines and support cell growth for co-culture and transmigration assays.

Millicell Single–well Standing InsertsDescription Pore size (µm) Device Size Qty/Pk Catalogue No.

PCF insert Isopore (Polycarbonate)

0.4 µm 24 well 50 PIHP012503.0 µm 24 well PITP012508.0 µm 24 well PI8P01250

12.0 µm 24 well PIXP012500.4 µm 6 well PIHP03050

Millicell Single–well Hanging InsertsDescription Pore size (µm) Device Size Qty/Pk Catalogue No.

PET Insert Polyethylene Terephthalate

0.4 µm 24 well 48 PIHT12R481.0 µm PIRP12R483.0 µm PISP12R485.0 µm PIMP12R488.0 µm PIEP12R48

MultiScreen-MIC SystemDescription Pore size (µm) Device Size Qty/Pk Catalogue No.

MultiScreen-MIC

* Includes a 96-well receiver plates housed in single-well trays, with lids. All parts are sterilized.

3.0 µm 96 well 10 MAMIC3S105.0 µm MAMIC5S108.0 µm MAMIC8S10

0

0.5

1.0

1.5

RFU

x 1

08

2.0

ReNcell CX MDA-MB-231

Cell Type

Laminin Cell Migration

Calcein-AM only

BSA

Lam

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Millicell µ-Angiogenesis Assay Kits(Catalogue No. MMA125 (Inhibition Kit) and Catalogue No. MMA130 (Activation Kit))

KEy ProDUCTS

FEATUrED ProDUCT

Description Catalogue No.

QCM Endothelial Cell Invasion Assay (24 well, colorimetric) ECM210

QCM Endothelial Cell Invasion Assay (24 well, fluorometric) ECM211

QCM Laminin Migration Assay (24-well, colorimetric) ECM220

QCM ECMatrix™ Cell Invasion Assay (24-well, 8 µm, colorimetric) ECM550

QCM Collagen Cell Invasion Assay (24-well, 8 µm, colorimetric) ECM551

QCM Collagen Cell Invasion Assay (24-well, 8 µm, fluorometric) ECM552

QCM ECMatrix Cell Invasion Assay (24-well, 8 µm, fluorometric) ECM554

QCM ECMatrix Cell Invasion Assay (96-well, 8 µm, fluorometric) ECM555

QCM Collagen Cell Invasion Assay (96-well, 8 µm, fluorometric) ECM556

QCM Tumor Cell Transendothelial Migration Assay (24 well, colorimetric) ECM558

QCM Haptotaxis Cell Invasion Series ECM580 series

ECL Cell Attachment Matrix 08-110

TNFa Growth Factor GF023

Millicell 24-well Receiver Tray with Lid PSMW010R5

Millicell Single-well Receiver Tray with Lid PSSW010R5

MultiScreen Single-well Culture Tray, clear, sterile MAMCS0110

MultiScreen 96-well Culture Tray clear, sterile MAMCS9610

• Easy-to-use, 15-well, microscope-like slide provides

complete optical visualization of meniscus-free wells

at low magnification

• Eliminates out-of-focus areas, in contrast to

96-well plates

• Compatible with multi-channel pipettes

and fixation reagents

• Uses 80% less reagents and cells for significant

cost savings

Studying how compounds affect angiogenesis, either to

promote or inhibit new tube formation, is critical to our

understanding of a number of diseases, especially tumor

progression. The Millicell µ-Angiogenesis activation and

inhibition kits provide a powerful platform for real-time

monitoring of changes in tubule formation with

unprecedented optical resolution.

8

Using surface receptors and adhesion molecules, cells respond to

signals from other cells and from the extracellular matrix (ECM).

These signals are translated into cell movements via tightly

regulated, site-specific protein complexes that link external signals

with the cytoskeleton. Recent studies have shown that

transmembrane signaling is often bidirectional, allowing intracellular

signals to be transmitted back to the surface proteins that control

movement along extracellular scaffolds. Cytoskeletal signaling

pathways control cell cycle, chromosomal organization, cell polarity,

nuclear dynamics, apoptosis, tumorigenicity, and more.

Cytoskeletal Signaling

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Signaling ProteinsCytoskeletal signaling complexes include G-protein complexes, focal adhesions, and adherens

junctions. The Rho family of small G-proteins transmit mechanical signals from the plasma

membrane. Rho family members Rac, Rho and Cdc42 regulate the assembly of actin-based

lamellipodia, stress fibers and filopodia, respectively. They also mediate polarity, proliferation,

apoptosis, membrane transport, and gene expression. Focal adhesions and adherens junctions are

membrane-associated complexes that link the cell exterior to the plasma membrane and the actin

cytoskeleton. Focal adhesions are key initiators of signaling in response to adhesion.

Phospho-FAK (Tyr397) STAr ELISA Kit (Catalogue No. 17-480)

Focal Adhesion Kinase (FAK) regulates cell migration via

integrin signaling. It autophosphorylates tyrosine 397

during adhesion and spreading, and is thought to modulate

focal adhesion turnover and adhesion-dependent growth

and survival. Phospho-FAK (Tyr397) is also correlated with

invasiveness of tumors.

ras GTPase Activation ELISA Kit (96 well) (Catalogue No. 17-424)

Ras GTPases regulate cell growth, proliferation,

differentiation, and survival. Inactive Ras is bound to GDP.

Active, GTP-bound Ras changes conformation, allowing it

to bind to effectors, including members of the Raf family,

the RalGDS family and PI3-kinase. The Ras activation ELISA

kit detects activated Ras using a chemiluminescent

substrate.

Lysates were treated with sodium vanadate (Na3VO

4) to enhance

phosphorylation. Phospho-FAK (Tyr397) STAR ELISA kit (17-480) was used

to determine that treated cells contained 4-12 times more phospho-FAK

(Tyr397) than did untreated cells.

Ras GTPase Activation ELISA Kit (17-424). In lysates of EGF-treated HeLa

cells, levels of activated Ras are elevated as compared to lysates of

untreated HeLa cells.

KEy ProDUCTS

Description Catalogue No.

Rac1/Cdc42 Activation Assay Kit 14-441

Focal Adhesion Pathway Explorer Antibody MiniPack 15-113

FAK STAR ELISA 17-479

Ras Activation ELISA Assay Kit 17-497

MILLIPLEX map 8-Plex Human Src Family Kinase Kit – Phosphoprotein 48-650

0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

HeLa L6 C2C12

Untreated

+Na3V04

FAK Tyr397 Phosphorylation Assay

Abs

orba

nce

at 4

50

nm

0

20Act

ivat

ion

(RLU

x 10

00

)

40

60

80

100

120

140

160

0 25

HeLa Cell Lysate (µg/well)

25

Assay Background

Unstimulated HeLa Cell Lysate

EGF Stimulated HeLa Cell Lysate

RasGTPase Activity Assay

10

The cytoskeleton consists of actin polymers, microtubules, and intermediate filaments. Actin

polymers, which mediate cellular movement and intracellular dynamics, are regulated by signaling

from many proteins, including those of the N-WASP family. Microtubules, directing movement of

subcellular components, are stabilized by Rho GTPases, microtubule-associated proteins, and

organization centers. IFs are a diverse family of structural proteins with equally diverse stabilizing

mechanisms. Molecular motor proteins use energy from nucleotide hydrolysis to move along actin

filaments or microtubules and include myosins, kinesins, and dyneins. These proteins mediate the

sliding of filaments, relative to one another, and transport organelles along cytoskeletal tracks.

Cytoskeleton

Actin Cytoskeleton and Focal Adhesion Staining Kit (Catalogue No. FAK100)

The organization of actin filaments and focal adhesions

reflects cell polarity, cell cycle state, differentiation status,

and other phenotypes. This staining kit is a sensitive

immunocytochemical tool to map the local orientation of

actin filaments and focal adhesions relative to the nucleus.

Acetyl-Cortactin Antibody (Catalogue No. 09-881)

Cortactin is a cytoskeleton protein that facilitates assembly

of cortical actin and is a prominent substrate of the

tyrosine kinase, Src. Recent studies show cortactin is

acetylated in vivo and is deacetylated by histone

deacetylase 6 (HDAC6). Cortactin acetylation regulates

actin-dependent cell motility by changing cortactin’s

affinity for filamentous actin. The acetylation level of

cortactin has also been correlated with tumor metastatic

potential.

Confocal fluorescence microscopy of focal adhesion and actin cytoskeleton

in COS-7 cells revealed with triple labeling using Phalloidin-TRITC

(Orange-red), Anti-Vinculin-FITC (Green), and DAPI (Blue).

Acetyl-Cortactin Antibody Staining. Anti-acetyl-Cortactin

(09-881) was used to

immunoprecipitate

acetylated cortactin

from untreated (lane 1)

or TSA/nicotinamide-

treated (lane 2) HeLa cells.

Immunoprecipitate was

resolved by SDS-PAGE and

transferred to PVDF. Blots

were probed with Anti-

acetyl-Cortactin.

Arrow indicates acetylated

cortactin (~ 85 kDa).

260

(kDa)1 2

160

11080

605040

30

20

1510

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KEy ProDUCTS

Description Catalogue No.

Anti-DYNLT1, clone 7A9F4H10 MAB1076

Anti-DYNLT3, clone 10A3C8A1 MAB1077

Anti-Actin, clone C4 MAB1501 (multiple)

Anti-Kinesin, heavy chain, clone H2 MAB1614

Anti-Myosin X AB1094

Description Catalogue No.

Anti-Dynein, Left - Right AB1961

Anti-Cortactin, clone 4F11 05-180

Anti-Myosin, heavy chain, clone A4.1025 05-716

Anti-mouse Tks4 09-260

Skeletal Myogenesis Assay KAA001

AXIS™ Axon Investigation System(Catalogue Nos. AX1510, AX50010, AX45005, AX45010, AX90010)

The AXIS platform is Millipore’s most advanced tool for the study of somas, neurites or synaptic formation. A slide-

mounted, microfluidic chamber system enables the deposition and culture of neural cells and spatially controlled

addition of growth factors, toxins, and other reagents. Neurite outgrowth is restricted to narrow, parallel channels,

and the resultant outgrowth or collapse behavior is easily observed under a microscope.

FEATUrED ProDUCT

Features , Benefits, Advantages:

• Organize, visualize, and

characterize neuronal cell

culture.

• Detect protein expression

with better spatial resolution.

• Isolate cell bodies from axons

through fluidics.

• Reduce time and expense

through optimized protocols

and QC validated products.

• Attain superior performance

over in-house protocols.

• Optically transparent, inert,

non-toxic, and nonflammable

polymer mold.

• Available in 150 µm, 450 µm,

900 µm, or 6-well format.

Overview

Steady-State Directional

Volume Mediated Hydrostatic Flow

MICROGROOVES

CHAMBER

Chemoattractant Gradient

Neuronal Culture+ Chemoattractant

12

Cell to cell and cell to extracellular matrix (ECM) adhesion enables

cell communication and movement, and is critical for the

development and maintenance of tissues. Adhesion is involved in

immune cell migration, metastasis of tumor cells, angiogenesis,

wound healing, and other processes. Changes in cell adhesion can be

the defining event in a wide range of diseases, including cancer,

osteoporosis, atherosclerosis, and arthritis. Adhesion between

tumor cells and their microenvironment involves integrins, matrix

metalloproteases (MMPs), and the ECM, and is of particular interest

as a targeted pathway for cancer therapy.

Adhesion

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Adhesion molecules that interact with the ECM enable the cell to move or maintain position. The

complex network of proteins and proteoglycans that make up the ECM can interact simultaneously

with multiple cell surface receptors. The most abundant ECM proteins are fibronectin, laminin, and

collagen. Many interactions with the ECM are mediated by integrins, which transduce bidirectional

signals controlling diverse processes, including cell growth, differentiation, migration, and apoptosis.

Cell to ECM Interactions

a Integrin-Mediated Cell Adhesion Array Kit (colorimetric) (Catalogue No. ECM530)

The majority of cell-ECM interactions occur via integrins, a large family of heterodimeric membrane glycoproteins consisting

of noncovalently associated a and b subunits. Integrin combinations can either recognize a single ECM ligand or bind several

different ECM proteins. This colorimetric integrin array kit is a cost effective, efficient method for identification of surface

integrins, and is a more rapid and quantitative than traditional methods.

a Integrin Adhesion Profile of Various Cell Lines

Several cell lines were incubated

with an array of individually-

coated a-integrin antibodies

on a 96-well plate. The graph

above shows differing integrin

expression levels for each

cell line. Adding or altering

experimental factors to this

type of test system allows one

to monitor the effects on the

adhesion signaling.

KEy ProDUCTS

Description Catalogue No.

Anti-Heparan Sulfate Proteoglycan, clone 7E12 MAB458

Anti-Integrin a6, clone NK1-GoH3 MAB1378

Anti-Heparan Sulfate Proteoglycan, Perlecan, clone Z7L6 MAB1948

Anti-Integrin aVb3, clone LM609 MAB1976 (multiple)

Anti-Laminin-g2, clone D4B5 MAB19562 (multiple)

Anti-Glypican-1, clone 4D1 MAB2600

Anti-Syndecan-1 AB8230

Other Integrin-Mediated Cell Adhesion Arrays ECM530 series

Integrin aVb3 purified protein CC1019

Integrin aVb5 purified protein CC1025

OD

at

56

0 n

m

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Illustrated by tissue patterning in multicellular organisms, the role of cell-cell adhesion molecules is

often to control migration, trafficking, organization, and final anchoring of specific cell types to their

niche. Major families of adhesion molecules include ephrins, cadherins (calcium dependent),

immunoglobulin-like adhesion molecules (CAMS), and selectins (carbohydrate binding). Cell-cell

adhesion molecules form boundaries and gradients that maintain tissue integrity in multicellular

organisms and facilitate cell-cell communication.

Cell to Cell Interactions

Endothelial Cell Adhesion Assay (Catalogue No. ECM645)

Both extravasation and intravasation are crucial steps

during tumor cell metastasis, and both processes require

tumor cell adhesion to the endothelial cells of blood vessel

walls. The Endothelial Cell Adhesion Assay measures the

affinity between circulating tumor cells and endothelial cells

in response to manipulation of activated or inactivated

endothelial cells.

Advantages include:

• Simple fluorometric screening format

• Endothelial activator and inhibitor controls

• Blocking antibodies included

ECM Cell Adhesion Array Kits (Catalogue Nos. ECM540 (colorimetric) and ECM545 (fluorometric))

Examine the adhesion, migration, and invasion of many diverse cell types on various ECM proteins with the ECM Cell Adhesion

Array Kits. The kits provide an ECM array microtiter plate with a homogenous detection format, allowing for large-scale

screening and quantitative comparison of multiple samples or cell lines. The kit is designed as a rapid (1-2 hours), cost

effective, efficient method for characterization of cell adhesion.

Adhesion of HL60 and THP-1 Cells to HUVECs measured by Endothelial

Adhesion Assay Kit (ECM645). Human Vascular Endothelial cells (HUVECs)

were cultured and treated with or without cycloheximide. The cells were

then activated with TNFa or IL-1b. Cycloheximide blocked > 90% of HL60

and THP-1 cell adhesion to the endothelium.

ECM Protein Cell Adhesion Fluorometric Array. The ECM adherence profile of

several cell lines is displayed across

various ECM proteins. Note that

each cell line has a unique adhesion

profile.

0

2

4

6

8

10

12

14

(+)Cycloheximide(+) Cytokine

(+)Cycloheximide(-) Cytokine

(-)Cycloheximide(+) Cytokine

(-)Cycloheximide(-) Cytokine

HL60 IL-1βTreatmentHL60 TNFαTreatment

THP-1 IL-1βTreatment

THP-1 TNFαTreatment

Adhesion Assay Conditions

Cyclohexamide Blocks Adhesion to Endothelial Cells

RFU

s x

10

4

ECM Protein Array Cell Adhesion Profile

0

10

20

30

40

50

RFU

s (4

85/

530

nm

x 10

00

) 60

Collagen I Collagen II Collagen IV Fibronectin Laminin

ECM Proteins

Tenascin Vitronectin Blank

HBK293

HT1080

NH3T3

CHO

MC3T3 E1

HUVECs

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KEy ProDUCTS

Description Catalogue No.

Anti-PECAM1, azide-free, clone 2H8 MAB1398Z

Anti-VE-cadherin, clone BV6 MAB1989

Anti-E-selectin, clone P2H3 MAB2150

Anti-Connexin 43, C-terminus, clone 4E6.2 MAB3067

Anti-E-cadherin, azide-free, clone 67A4 MAB3199Z

Anti-MCAM (CD146), azide-free, clone P1H12 MAB16985

Anti-Connexin 45, near C-terminus, cytoplasmic AB1745

Description Catalogue No.

E-selectin ELISA Kit ECM330

ICAM-1 ELISA Kit ECM335

VCAM ELISA Kit ECM340

QCM Leukocyte Transendothelial ECM557

Migration Assay (24 assays, colorimetric)

QCM Tumor Cell Transendothelial ECM560

Migration Assay (24 assays, fluorometric)

QCM Leukocyte Transendothelial Cell Migration Assay(Catalogue No. ECM559)

In order for leukocytes to migrate to distant locations of infection or inflammation, they must circulate in the

bloodstream, then migrate out of a blood vessel (extravasation). The penetration of circulating leukocytes into the

endothelium is a crucial step in the process. This transendothelial cell migration assay enables quantitative analysis

of the ability of leukocytes to invade the endothelium.

Features , Benefits, Advantages:

• Statistical relevant analysis of multiple samples

• Consistent, convenient solution to a complex assay

• Fluorescent labeling technique returns highly specific results

FEATUrED ProDUCT

Transendothelial Migration of Leukocytes. Migration of HL-60 cells through stimulated (TNFa) and unstimulated endothelial cell layer over 6

hours, using fetal bovine serum (FBS) as a chemoattractant. The data in Figure 1 indicates that a greater number of leukocytes invade an activated

endothelial cell system over unstimulated conditions. Note, however, over an extended period of time (18 hours), HL-60 cells eventually migrate

towards an FBS chemoattractant as outlined in Figure 2.. Additionally, an inactive endothelial layer does not provide the appropriate signaling

cascade for intravasation of leukocytes. HUVECs were grown to confluency on cell culture inserts provided.

1

2

4

3

0

1

2

3

No Treatment

TNFα

no s

erum

10%

FB

SO.D

. 570 n

m

no s

erum

10%

FB

S

0

ControlTNFα

10%

FB

SRFU

x 1

000

10%

FB

S 321

4

98765

0

Control10% FBS

RFU

x 1

000

0

1

2

3

HUVEC

HUVEC+HT-1080

no s

erum

10%

FB

SO.D

. 570 n

m

no s

erum

10%

FB

S

1

2

4

3

0

1

2

3

No Treatment

TNFα

no s

erum

10%

FB

SO.D

. 570 n

m

no s

erum

10%

FB

S

0

ControlTNFα

10%

FB

SRFU

x 1

000

10%

FB

S 321

4

98765

0

Control10% FBS

RFU

x 1

000

0

1

2

3

HUVEC

HUVEC+HT-1080

no s

erum

10%

FB

SO.D

. 570 n

m

no s

erum

10%

FB

S

Figure 1. Figure 2.

16

The extracellular matrix (ECM) surrounds and supports the cells

within living systems. The most prominent class of ECM proteins

consists of structural proteins of the collagen and elastin families.

Collagen fibers strengthen and organize the matrix; elastin fibers

provide flexibility and resilience. Adhesive proteins such as

fibronectin, laminin, and tenascin enable cell attachment and form

cross-links within the matrix. In addition, numerous proteoglycans

and heparan sulfate-containing proteins form the hydrated, gel-like

mixture that helps stabilize the matrix within its aqueous

environment.

Extracellular Matrix and Associated Proteases

17

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Extracellular matrix (ECM) proteins are produced intracellularly and are subsequently secreted into

the surrounding cellular medium, actively regulating a diverse range of cell functions including cell

adhesion, differentiation, proliferation, migration, invasion, and survival.

ECM Proteins

ECM Cell Culture optimization Arrays (96-well, fluorometric) (Catalogue No. ECM546)

Adding ECM proteins to in vitro cell cultures can promote

cellular adhesion while maintaining cell viability and

maximizing cell proliferation for downstream cell-based

applications. The ECM Cell Culture Optimization Array

provides a multiparametric assay that quickly identifies

ECM protein(s) and pinpoints optimal concetrations that

best promote cell-type specific adhesion for a particular

cell type.

ECM Proteins and Antibodies Different cell types have different affinities for various ECM

proteins, depending on which cell surface proteins are

expressed. Millipore offers a wide variety of ECM proteins

and antibodies to meet the Individual growth needs of any

cell line.

Mouse anti-Fibronectin, CS-1.Staining of normal

human gut mucosa.

(Cat. No. MAB1939)

ReNcell VM adhesion profiling against ECMs. Human neural stem cells,

ReNcell VM, were seeded on the ECM Cell Culture Optimization Array at

105 cells per well for 2 hours at 37 °C. Cell adhesion levels were measured

by crystal violet staining and analyzed by spectrometer. Each data set

represents three replicates.

0.0

0.5

1.0

1.5

Opt

ical

Den

sity

(O

D 5

70

nm

)

ECM (µg/mL)

ReNcell VM

0 5 10 15 20 25

LN

FN

COL

VN

KEy ProDUCTS

Description Catalogue No.

QuantiMatrix Human Fibronectin ELISA ECM300

QuantiMatrix Human Laminin ELISA ECM310

3D Collagen Culture Kit ECM675

Osteogenesis Assay ECM810

In Vitro Osteogenesis Quantitative Assay ECM815

Collagen Type I, rat tail 08-115

Mouse Laminin CC095

Description Catalogue No.

Proteoglycan CC117

Human Plasma Fibronectin Purified Protein FC010 (multiple)

Soluble RANK Ligand (sRANKL), recombinant human GF091

Wnt-3a, recombinant mouse GF160

Mouse Bone Metabolism Panel 1A MBN1A-41K

Rat Bone Metabolism Panel 1 RBN1-31K

Mesenchymal Stem Cell Osteogenesis Kit SCR028

18

Matrix metalloproteinases (MMPs) are secreted or transmembrane proteolytic endopeptidases that

process and degrade extracellular matrix proteins. MMPs play critical roles in many normal growth

and developmental aspects of tissue remodeling, wound healing, and angiogenesis. In a pathological

context, MMPs are associated with cell migration, invasion, arthritis, and cancer tumor progression.

Once activated, the MMPs are subject to inhibition by the tissue inhibitors of metalloproteinases

(TIMPs) that bind MMPs non-covalently.

Metalloproteinases

MT1-MMP Antibody (Catalogue No. AB6004)

Most MMPs are secreted as inactive proproteins which are

activated when cleaved by extracellular proteases. However,

MT1-MMP (MMP-14) is a member of the membrane-type

subfamily. Each member of this subfamily contains a

potential transmembrane domain suggesting that they are

expressed at the cell surface rather than secreted.

MT1-MMP mediates pericellular proteolysis of ECM

components and is important for matrix remodeling. This

protein also activates MMP2 protein, and this activity may

be involved in tumor invasion.

GM6001 MMP Inhibitor (Catalogue No. CC1000, CC1010, CC1100)

GM6001 (Ilomastat or Galardin) is a potent broad-spectrum

hydroxamate inhibitor of matrix metalloproteinases (MMPs)

that can be used in both in vitro and in vivo applications to

assess the biological effects of perturbing MMP activity.

GM6001 inhibits MMP-2 activity at concentrations greater than 0.1 nM

0

10

MM

P-2

Act

ivit

y (%

)

20

30

40

50

60

70

80

90

100

0 0.01 0.1 1.0 10 100

GM6001 (nM)

Dose-Response for GM6001 effect on MMP-2 Activity

Immunohistochemcial staining of MT1-MMP in paraffin-embedded colorectal

carcinoma tissue using anti-MT1-MMP, hinge region (AB6004). Pattern

depicts a cytoplasmic to diffuse membrane staining of malignant cells.

19

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MILLIPLEX® map Human Bone Multiplex Panel(Catalogue No. HBN1B-51K)

Bone metabolism balances bone deposition with bone resorption. While osteoblasts and osteocytes control bone

deposition, osteoclasts effect bone resorption. To maintain this metabolic balance, cells rely on signaling involving

hormones and cytokines. Disruption of bone metabolism results in diseases like osteoporosis, osteoarthritis,

rheumatoid arthritis, chronic kidney disease, and bone metastases. Quickly assess the status of bone metabolism in

human research samples using the MILLIPLEX map Human Bone Panels, which enable multiplex detection of several

highly relevant analytes in either serum matrix or cell/tissue lysates.

Features , Benefits, Advantages:

• Luminex xMAP technology dramatically increases speed, sensitivity, and productivity

• Negligible cross-reactivity between analytes or antibodies for high accuracy

• Reproducible analysis using as little as 25 µL of sample per well

FEATUrED ProDUCT

MILLIPLEX map Human Bone Panel 1B (Catalogue No.

HBN1B-51K) quantitatively measures bone biomarkers in

cell and tissue lysates over a wide linear range.

KEy ProDUCTS

Description Catalogue No.

Anti-MT1-MMP, clone 3G4.2 MAB1767

Anti-MT1-MMP AB8345

Anti-TIMP-3, C-terminus AB6000

Anti-MMP-1, hinge region AB6002

Anti-MMP-2, hinge region AB6003

Anti-TIMP-1, C-terminus AB6007

Description Catalogue No.

Anti-MMP-9 AB19016

GM6001 MMP Inhibitor CC1010 (multiple)

Human MMP Panel 1 HMMP1-55K

Human MMP Panel 2 HMMP2-55K

Human TIMP Panel 1 HTIMP1-54K

Human TIMP Panel 2 HTIMP2-54K

100,000

Human Bone Panel 1AStandard Curves in Serum Matrix

10,000

1,000

100

10

10 10 10

0

1,00

0

10,000

100,00

0

1,00

0,00

0

Med

ian

Fluo

resc

ence

Inte

nsit

y (M

FI)

Concentration (pg/mL)

Human RANKL Single PlexStandard Curve in Assay Buffer

10,000

1,000

100

10

10 10 10

0

1,00

0

10,000

100,00

0

Med

ian

Fluo

resc

ence

Inte

nsit

y (M

FI)

Concentration (pg/mL)

SerumMatrix

100,000

Human Bone Panel 1BStandard Curves in Assay Buffer

10,000

1,000

100

10

10 10 10

0

1,00

0

10,000

100,00

0

1,00

0,00

0

Med

ian

Fluo

resc

ence

Inte

nsit

y (M

FI)

Concentration (pg/mL)

1

OPG

PTH

Leptin

ACTH

Osteopontin

Insulin

Osteocalcin

IL-1β

Adiponectin

IL-6

TNFα

100,000

Human Bone Panel 1AStandard Curves in Serum Matrix

10,000

1,000

100

10

10 10 10

0

1,00

0

10,000

100,00

0

1,00

0,00

0

Med

ian

Fluo

resc

ence

Inte

nsit

y (M

FI)

Concentration (pg/mL)

Human RANKL Single PlexStandard Curve in Assay Buffer

10,000

1,000

100

10

10 10 10

0

1,00

0

10,000

100,00

0

Med

ian

Fluo

resc

ence

Inte

nsit

y (M

FI)

Concentration (pg/mL)

SerumMatrix

100,000

Human Bone Panel 1BStandard Curves in Assay Buffer

10,000

1,000

100

10

10 10 10

0

1,00

0

10,000

100,00

0

1,00

0,00

0

Med

ian

Fluo

resc

ence

Inte

nsit

y (M

FI)

Concentration (pg/mL)

1

OPG

PTH

Leptin

ACTH

Osteopontin

Insulin

Osteocalcin

IL-1β

Adiponectin

IL-6

TNFα

To PLACE An orDEr or rECEIVE TECHnICAL ASSISTAnCE In the U.S. and Canada, call toll-free 1 800-Millipore (1-800-645-5476)

In Europe, please call Customer Service:

France: 0825.045.645

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Italy: 848.845.645

English UK: 0870.900.46.45

For other countries across Europe and the world,

please visit www.millipore.com/offices.

For Technical Service, please visit www.millipore.com/techservice.

Your Source for Cell Structure ResearchWe at Millipore are excited that the latest findings in developmental biology, stem cell research,

neuroscience, and oncology have placed cell structure and movement at the crossroads of so

many diverse research areas. As the only life science supplier to be a steady partner to all these

fields, we are committed to providing the most complete solutions for advancing your research.

For more information, please visit our website at www.millipore.com/cellstructure.

Millipore, Upstate, Chemicon, guava, MILLIPLEX, Millicell, Multiscreen, and Advancing Life Science Together are registered trademarks. The M Mark, Flowcellect, QCM, AXIS, ECMatrix, and QuantiMatrix are trademarks of Millipore Corporation. Luminex and xMAP are registered trademarks of Luminex Corporation. ReNcell is a trademark of ReNeuron Group PLC. Lit. No. PB2932EN00 Printed in U.S.A. 03/10 LS-SBU-10-02891 © 2010 Millipore Corporation, Billerica, MA 01821 U.S.A. All rights reserved.

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