View
291
Download
7
Category
Preview:
DESCRIPTION
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. Specìcally, inhibiting tumor invasion by blocking tumor cell chemotaxis has been a major focus of research.
Citation preview
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.
3
ww
w.m
illipore.com/cellstructure
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
5
ww
w.m
illipore.com/cellstructure
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
7
ww
w.m
illipore.com/cellstructure
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
9
ww
w.m
illipore.com/cellstructure
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
11
ww
w.m
illipore.com/cellstructure
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
13
ww
w.m
illipore.com/cellstructure
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
14
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
15
ww
w.m
illipore.com/cellstructure
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
ww
w.m
illipore.com/cellstructure
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
ww
w.m
illipore.com/cellstructure
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
Spain: 901.516.645 Option 1
Germany: 01805.045.645
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.
www.millipore.com/cellstructure
Recommended