CancerComprehensive Solutions from Hallmarks to Biomarkers

Data SheetProduct Selection Guide

Hallmarks of

Cancer

Self-sufficiency inproliferation signals

Insensitivity to

proliferation

inhibiting signals

Evading apoptosis

Tiss

ue in

vasio

n

& met

asta

sis

Limitl

ess r

eplic

ative

pote

ntial

Sustained angiogenesis

Metabolic

reprogramming

Escape from

imm

une control

ATPSTOPTOP

As a tools provider and partner in research, EMD Millipore is committed to the advancement 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

extensive technical support.

Platforms and Technologies

Antibodies and ImmunoassaysEMD Millipore offers an extensive, focused portfolio of antibodies and immunoassays. With the expertise of

Upstate® and Chemicon®, EMD Millipore provides validated products with breadth and depth in major

research areas backed by excellent service and support.

Cell-Based Assays and Quantitative Cell ImagingEMD Millipore 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. EMD

Millipore also offers an array of assays for high-content, multiparametric cell imaging, enabling identifica-

tion 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. EMD Milli-

pore 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, EMD Millipore’s

flexible and customizable assays are exhaustively tested and qualified for sensitivity, specificity,

reproducibility and wide dynamic range.

Calbiochem® CompoundsCalbiochem provides high quality inhibitors, biochemicals, antibodies, proteins, and kits that have been cited

in thousands of peer-reviewed publications. Small-molecule compounds, including inhibitors, activators, and

other pathway modulators, are critical tools for researchers studying cell signaling and other intracellular

mechanisms that control cell fate, function and phenotype. From libraries and pathway panels to individual

reagents, the Calbiochem line of products offers the widest and most cited selection of inhibitors and

activators worldwide.

ServicesEMD Millipore advances drug discovery and evaluation by providing products and services to complement

your work and help you achieve results faster than ever before. We offer a suite of products that span the

drug discovery pipeline from target identification to clinical studies. Our expert team of scientists and

engineers understands the complexity of your discovery and development and can support you in these

challenges.

3

CoMPrEHEnSIvE SoLutIonS FroM HALLMArkS to BIoMArkErS Our understanding of cancer is rooted in identifying the

phenotypic differences between cancer cells and

corresponding normal cells of the same lineage. Although the

recent, huge output of comparative genomic, proteomic, and

epigenomic data comparing tumor and non-tumor has yet to

be fully mined, the data point to eight key traits, shared by

most tumor types, that drive disease progression. These are

listed in the table of contents (right).

These “hallmarks” of cancer, an update to the original list

of Hanahan and Weinberg1, are important—not only because

they represent opportunities for therapeutic intervention, but

also because they are opportunities to use tumors as models

to decipher the signaling pathways underlying both normal and

diseased cellular processes. As a result, oncology research not

only impacts cancer drug discovery, but also advances

understanding of all of biology, from developmental

mechanisms to aging.

Although it is tempting to view these eight hallmarks as a

linear path ending in metastatic cancer, challenges such as

tumor heterogeneity, individual polymorphisms, nonparallelism

in model systems, and inability to distinguish cell-intrinsic from

cell-extrinsic factors require an unbiased, multidisciplinary,

cross-platform approach to cancer research.

Recognizing both the tremendous opportunities and the

challenges facing cancer research, EMD Millipore has been

dedicated to developing and refining technologies for the

study of cancer. With EMD Millipore’s comprehensive portfolio,

including the Upstate, Chemicon, and Calbiochem brands of

reagents and antibodies, researchers can count on

dependable, high quality solutions for analyzing all the

hallmarks of cancer.

Reference

1. Hanahan D., Weinberg R.A. (2000) The Hallmarks of Cancer. Cell. 100:57-70.

FEAturED ProDuCtS• MILLIPLEX map EpiQuant EGFR Pathway 6

Magnetic Bead Panel

• Calbiochem InhibitorSelect 96-well Protein 6

Kinase Inhibitor Library I

• FlowCellect Bivariate Cell Cycle Kit for DNA Replication 10

• FlowCellect MitoDamage Kit 14

• Calbiochem InhibitorSelect Akt/PI 3-K/mTOR 15

Signaling Pathway Panel

• H2A.X Phosphorylation & p53 DNA Damage Assay 15

• Phospho-Histone H3 (Ser10) and Cyclin B1 18

QCI /HCA Assay

• In Vitro Vascular Permeability Assay 19

• MILLIPLEX map Human Circulating Cancer 27

Biomarker Panel 1

tABLE oF ContEntS

Self-sufficiency in proliferation signals 4

Insensitivity to proliferation inhibiting signals 9

Evading apoptosis 13

Limitless replicative potential 17

Sustained angiogenesis 19

Metabolic reprogramming 23

Escape from immune control 26

Tissue invasion and metastasis 28

ATP

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Introducing EMD MilliporeThe acquisition of Millipore by Merck KGaA brings the

expertise and complementary capabilities of two

companies with a history of providing superior products

and solutions to the Life Science industry. As EMD

Millipore, we are intent on being your world-class Life

Science partner, driving innovation through strong

customer relationships, significant investment in R&D and

the contribution of our employees. This Cancer Product

Selection Guide is the first integrated presentation of our

comprehensive range of solutions to support cancer-

related research.

4

Anti-mtor, clone 2ID8.2 (Catalogue No. 05-1592)

mTOR affects cell proliferation and survival primarily by

phosphorylating Akt, an effector of the PI3 kinase pathway. In

addition, mTOR regulates cell growth and autophagy. The mTOR

pathway is frequently dysregulated in cancer. Validated for

Western blotting and immunocytochemistry, anti-mTOR is one

of our numerous antibodies for studying self-sustained

proliferation, including key receptors such as EGFR, ErbB2, Met,

VEGFR, and IR and downstream targets such as Ras, PI3-Kinase,

Akt, Mek, and Erk.

PI3 kinase Activity/Inhibitor ELISA (Catalogue No. 17-493)

The discovery of tumor-specific mutations in PI3 kinase (PI3K)

genes has shifted research focus on PI3K from basic

biochemistry to target validation and drug development. These

mutations single out class I PI3K as an important contributor

to oncogenesis. Most human cancers also show activated PI3K

signaling. Easily evaluate PI3K activation and inhibition with this

competitive ELISA kit. The PI3 Kinase Activity/Inhibitor Assay

enables fast and sensitive quantitation of activity of the four

class I PI3 kinases (p110 a, b, d, g).

Self-Sufficiency in Proliferation Signals

Confocal immunofluorescence analysis of NIH 3T3 cells using anti-mTOR

(05-1592) (red) shows diffuse cytoplasmic distribution. Actin filaments have

been labeled with Alexa Fluor 488-Phalloidin (green). Nuclei are stained with

DAPI (blue).

Results from the PI3K ELISA kit demonstrate inhibition via isoform-specific

and general class I inhibitors.

Normal cell proliferation is commonly regulated by cell signaling pathways that lead to reversible

modification of existing proteins and phospholipids. These modifications, which include

phosphorylation, methylation, acetylation, ubiquitination, and hydroxylation, function in

combination to control individual proteins or multi-protein pathways. While normal cells require

extracellular cues for cell division, cancer cells possess mutations, often due to genetic

instability, that render their proliferation independent of these cues. Rapid proliferation, in

turn, provides more opportunities for tumor cells to acquire mutations favoring tumor

progression.

Selectivity and Profiling AgainstPI3 Kinase Isotypes

0

20

40

60

80

100

120LY294002

Wortmannin

PI-103

TGX-221

AS-252424

% C

ontr

ol

p110α p110β p110δ p110γ

Selectivity and Profiling AgainstPI3 Kinase Isotypes

0

20

40

60

80

100

120LY294002

Wortmannin

PI-103

TGX-221

AS-252424

% C

ontr

ol

p110α p110β p110δ p110γ

Selectivity and Profiling AgainstPI3 Kinase Isotypes

0

20

40

60

80

100

120LY294002

Wortmannin

PI-103

TGX-221

AS-252424

% C

ontr

ol

p110α p110β p110δ p110γ

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STOPTOP

5

ras Activation ELISA Assay (Catalogue No. 17-497)

Oncogenic mutations resulting in constitutively active Ras are

correlated with tumor development in a variety of tissues.

Therefore, quantitatively measuring stimulated Ras is

important for the study of oncogenic pathways. This kit

detects activated Ras through selective binding of immobilized

Raf-1 in the presence of GTP. Using a monoclonal anti-Ras

antibody, detection of Ras isoforms across three different

species enables simple quantitative analysis and is amenable to

high throughput environments. Individual components are also

available.

FlowCellect PI3k/MAPk Dual Pathway Activation and Cancer Marker Detection kit (Catalogue No. FCCS025100)

The two kinases PI3K and MAPK often mediate aberrant cell proliferation, and recent research suggests cross-talk among their

downstream effectors. This flow cytometry kit enables easy analysis of the role of cross-talk in proliferation by providing three

fully validated and optimized antibodies to measure specific cell signaling events along both pathways. The kit uses directly labeled

antibodies against phospho-Akt and phospho-ERK1/2 to analyze signaling activation and cross-talk, plus a Ki-67 marker to identify

the proliferative fraction.

Elevated levels of activated Ras are easily detected in HeLa cells

stimulated with EGF versus unstimulated cells.

HEK293 cells were stimulated by insulin (B, E) or PMA (C, F) and simultaneously stained with antibodies against phospho-Akt and phospho-ERK1/2. The cross-talk

between the PI3K and MAPK signaling pathways is demonstrated by the sharp decrease in ERK phosphorylation after five minutes of insulin stimulation.

1 x 106

0

2 x 106

3 x 106

4 x 106

5 x 106

6 x 106

7 x 106

1040.97% 0.47%

0.30%

103

102

101

100

100 101 102 103 104

pAKT-Alexa 488

pER

K-P

E

98.26%

1040.78% 61.94%

30.37%

103

102

101

100

100 101 102 103 104

pAKT-Alexa 488

pER

K-P

E

6.91%

104 100%90%80%70%60%50%40%30%20%10%

0%pAkt pErk

Untreated100 nM Insulin 3 min100 µg/mL PMA 3 min

Ki67

30.30% 60.06%

1.28%

103

102

101

100

100 101 102 103 104

pAKT-Alexa 488

pER

K-P

E

8.35%

104 1.04% 0.31%

0.18%

103

102

101

100

100 101 102 103 104

pAKT-Alexa 488

A. Untreated B. Insulin Treated C. PMA Treated

D. Untreated E. Insulin Treated F. PMA Treated

HEK293

HEK293

3-M

inut

e Sti

mul

atio

n5

-Min

ute

Sti

mul

atio

n

pER

K-P

E

98.47%

104 0.72% 15.46%

75.15%

103

102

101

100

100 101 102 103 104

pAKT-Alexa 488

pER

K-P

E

8.67%

104 34.29% 49.52%

3.46%

103

102

101

100

100 101 102 103 104

pAKT-Alexa 488

pER

K-P

E

12.73%

100%90%80%70%60%50%40%30%20%10%

0%pAkt pErk Ki67

6

InhibitorSelect 96-Well Protein Kinase Inhibitor

Libraries I & II (160 inhibitors; Cat. Nos.

539744* and 539745*) were screened for

influence on proliferation and survival of mouse

neural stem cells (mNS) in a cell viability assay

under 4 conditions:

(A) No GFs – No Growth Factors (to identify

survival/proliferation factors)

(B) Sub EGF – Sub-optimal EGF (to identify

inhibitors/potentiators)

20 pg/mL EGF

(C) Sub FGF2 – Sub-optimal FGF2 (to identify

inhibitors/potentiators)

500 pg/mL FGF2

(D) Max GFs – Maximal EGF + FGF2 (to identify

inhibitors/potentiators)

20 ng/mL EGF + 20 ng/mL FGF2

The presence of inhibitor K-252a, Nocardiopsis

sp. (Cat. No. 420297) alone in the culture

medium resulted in a 10-fold mNS cell viability.

Data courtesy of Donna McLaren, Stem Cell Sciences, Cambridge, UK

Phosphorylated EGFR pathway proteins, as well as total EGFR, were

simultaneously detected in A431 cells treated with 1000, 333, 111, 37,12.3

and 0 ng/mL EGF. Lysates were collected after 5 minutes EGF stimulation.

Values are internally normalized utilizing the TAFII68 loading control.

MILLIPLEX map EpiQuant EGFr Pathway Magnetic Bead Panel (Catalogue No. MPEQMAG-110K)

Epidermal growth factor receptors (EGFR) play crucial roles

in regulating cell proliferation, differentiation, motility, and

apoptosis, contributing to cancer and other pathological

processes. EpiQuant technology enables absolute

quantitation of the phosphorylation status of EGFR family

members and related signal transduction proteins, providing

a thorough understanding of this signaling pathway. By

detecting multiple analytes simultaneously, this assay

conserves sample and increases accuracy. The magnetic

bead format of the assay provides additional convenience,

with walk-away washing options, and increased consistency,

with lower coefficients of variation.

Calbiochem InhibitorSelect 96-Well Protein kinase Inhibitor Library I (EMD Chemicals Catalogue No. 539744*)

This panel of compounds consists of 80, well-characterized protein kinase inhibitors, the majority of which are cell-

permeable and ATP-competitive. The library is useful for cancer signaling pathway analysis, cell-based assays, target

identification in drug discovery, screening new protein kinases, and other related applications. It is supplied with a CD

containing comprehensive documentation for each inhibitor.

[EGF] (ng/ mL)

[Ana

lyte

] (p

M)

EpiQuant EGFR Panel: EGF Dose Response

EGFR (pY1110/1125)

ERK1/2 (pY204/187)

1

10

100

1,000

10,000

1 10 100 1,000 10,000

EGFR (pY1069/1092)

Shc (pY349/350)

ErbB3 (pY1197/1307)

0

2

4

6

8

10

12No GFs

Sub EGF

Sub FGF2

Max GFs

Mouse Neural Stem Cell Viability

SU

6656

GSK-3

Inhi

bito

r XI

II

Isog

ranu

lati

mid

e

IC261

IKK-2

Inhi

bito

r IV

Indi

rubi

n D

eriv

ativ

e E8

04

JNK In

hibi

tor

II

JNK In

hibi

tor,

Neg

ativ

e C

ontr

ol

JNK In

hibi

tor

V

JNK In

hibi

tor

IX

MK2a

Inhi

bito

r

JNK In

hibi

tor

VIII

K-2

52a,

Noc

ardi

opsi

s sp

.

Ken

paul

lone

KN

-93

MEK

Inhi

bito

r I

MEK

Inhi

bito

r II

MEK

1/2

Inhi

bito

r

MN

K1

Inhi

bito

r

NK-k

B A

ctiv

atio

n In

hibi

tor

Fold

cha

nge

rela

tive

to

DM

SO

Con

trol

Gro

wth

Fac

tor

Bac

kgro

und

7

Lead Discovery Profiling Services

GPCrProfiler ServiceGPCRProfiler is the first complete cell-based functional

platform that uses a common validated readout for over

155 GPCRs. The foundation of GPCRProfiler is EMD

Millipore’s ChemiScreen GPCR stable cell lines that are used

for real-time calcium flux assays to rapidly, reliably and

reproducibly screen and profile compounds. Using one

platform allows ligands to be screened with identical buffer

conditions and incubation times for the entire spectrum of

GPCRs for easy analysis and comparison.

Quickly implement screening in a new disease area or

target receptor, or increase your current screening

capacity with GPCRProfiler services. Our unbiased profiling

screens can determine if drugs are full or partial agonists

or antagonists. We also perform dose-response assays to

determine EC50 values for agonists and IC

50 values for

antagonists. You will typically receive results within 1-3

weeks of compound submission, in a thorough and intuitive

report. GPCRProfiler gives you the flexibility to choose from

1 to ≥155 different receptors to screen. Or, choose

between 15 different service panels, including more than

60 distinct ligand families, 2 safety panels and 11 disease-

focused panels.

kinase & Phosphatase Profiling ServicesIn 2000, the KinaseProfiler service developed by Upstate,

now a part of EMD Millipore, brought selectivity profiling to

kinase drug discovery researchers. Today, the

KinaseProfiler panel includes almost 300 protein and lipid

kinases, 21 phosphatases and a complementary suite of

secondary assays, forming the most diverse, disease-

relevant panel available commercially. As the partner of

choice for kinase screening, we provide validated data using

the robust and reliable radiometric kinase assay trusted by

the world’s leading pharmaceutical companies.

SignalProfiler ServicesProviding fresh insights to the cellular activity of

compounds, SignalProfiler service employs MILLIPLEX

MAPmate cell signaling kits to profile samples against a

panel of 90+ modified and total proteins. SignalProfiler can

be used to measure the effectiveness of kinase inhibitors

and GPCR ligands by measuring changes in signaling

cascades in relevant cell backgrounds. In addition, early

signaling events leading to cellular toxicity can be studied

to help assess potential compound liabilities in early stages

of drug development.

tECHnoLoGY HIGHLIGHt

Mutations of many receptor tyrosine kinases, G protein-coupled receptors, and intracellular kinases are responsible for

uncoupling the requirement for external proliferation cues in cancer cells. Many small molecule GPCR and kinase inhibitors

have been successfully been developed as anti-tumorigenic drugs. EMD Millipore’s Lead Discovery Services are well suited to

extensive profiling of these drugs against a broad range of targets to reveal interactions that may have additional

therapeutic value or present adverse reactions.

Visual display using EMD Millipore’s DART™ (Data Analysis and

Reporting Tool) of the inhibitory activity of the anti-cancer agent

imatinib (Gleevec®, Novartis) which was functionally profiled against

a large portion of the kinome using KinaseProfiler service.

8

kEY ProDuCtS - ProLIFErAtIon SIGnALInG

Antibodies and Proteins

Description Catalogue No.

Anti-EGFR, neutralizing, clone LA1 05-101

Anti-phospho-erbB-2/HER-2 (Tyr1248) 06-229

Anti-Ras, (K-, H-, N-), clone 9A11.2 05-1072

Anti-Ras-related protein Ral-A 07-2132

EGFR, active, purified kinase 14-531

Assays

Erk 1/2 (Thr202/Tyr204)/Thr185/Tyr187) 17-442

Dual Detect CELISA

PIP3 Quantification TR-FRET Assay 17-494

c-ErbB2/c-Neu Rapid Format ELISA Kit QIA10*

Description Catalogue No.

Raf-1 Kinase Assay Kit, 17-360

Chemiluminescence Detection

MILLIPLEX map EpiQuant EGFR Pathway MPEQMG-110K-PX22

Magnetic Bead 22-Plex Premix

MILLIPLEX map MAPK/SAPK Cell Signaling 10-Plex 48-660

FlowCellect MAPK Activation Dual Detection kit FCCS025106

FlowCellect PI3K Activation Dual Detection Kit FCCS025105

FlowCellect EGFR/MAPK Pathway Activation FCCS025101

Detection Kit

K-LISA™ mTOR (Recombinant) Activity Kit CBA104*

PI3-Kinase HTRF Screening Assay 33-016

Phospho-ERK1/2 (Thr185/Tyr187) QCI / HCA Assay Kit HCS230

Compound Libraries

InhibitorSelect 96-Well Protein Kinase Inhibitor Library II 539745*

InhibitorSelect 96-Well Protein Kinase Inhibitor Library III 539746*

InhibitorSelect 384-Well Protein Kinase Inhibitor Library I 539743*

StemSelect® Small Molecule Regulators 384-Well Library I 569744*

*Available from EMD Chemicals Inc.

For a complete listing of cancer-related products from Millipore, please visit www.millipore.com/cancer.

For a complete listing of cancer-related products from EMD Chemicals, please visit www.emdbiosciences.com.

9

Anti-Cyclin B1, clone GnS3 (8A5D12) (Catalogue No. 05-373)

Cyclin B1 triggers mitosis by binding and activating cdc2

kinase, which phosphorylates multiple protein targets.

Validated for immunoprecipitation and Western blotting, this

monoclonal antibody complements EMD Millipore’s huge

portfolio of transcription factor antibodies, including other

important cell cycle regulators, including Rb, Wee1, and CDCs.

EMD Millipore also offers numerous Antibody Minipacks

supporting various cell cycle stages and DNA repair pathways

that enable you to explore more for less.

Akt (Ser473) Dual Detect CELISA Assay kit (Fluorogenic Detection) (Catalogue No. 17-444)

The Ser/Thr kinase Akt (PKB), when phosphorylated on Ser473

by the mTOR complex, becomes active and phosphorylates

targets in multiple pathways including cell survival, cell cycle

control, cell growth, and cell metabolism. Akt pathways are

frequently dysregulated in cancer. The Dual Detect CELISA is

an efficient and sensitive assay for simultaneous quantification

of phosphorylated Akt (Ser473) and total Akt in whole cells.

Non-stimulated A431 cell lysate

was resolved by electrophoresis,

transferred to nitrocellulose

and probed with anti-Cyclin B1

(05-373, 0.5 µg/mL). Proteins

were visualized using a goat

anti-mouse secondary antibody

conjugated to HRP and a

chemiluminescence detection

system. Arrow indicates Cyclin

B1 (~58 kDa).

NIH3T3 cells were seeded at 1x104 cells per well, cultured for 24 hr, serum

starved 16 hr, and stimulated with 50 ng/mL of PDGF for 0, 1, 5, 15, or 30

minutes. Total and phospho Akt levels were determined using Akt (Ser473)

Dual Detect CELISA Kit (17-444). Phosphorylated Akt levels are normalized

to the total Akt. Inset shows the corresponding Western blot.

97

66

45

00

0 1 5 15 30 minTotal-AKTPhospho-AKT (s473)

1 5 15 30

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Rel

ativ

e Pho

spho

-Akt

(Ser

47

3)

(Nor

mal

ized

by

Tota

l Akt

)

Time (minutes)

NIH3T3 cells treated with 50 ng/mL PDGF

Insensitivity to Proliferation Inhibiting SignalsTo maintain normal cellular homeostasis, proliferation inhibiting signals, including soluble

inhibitors and inhibitors residing on the surface of nearby cells, must be received and

transmitted through intracellular pathways. Adaptation through mutation may render tumor

cells resistant to these proliferation inhibiting signals, enabling them to overcome cell cycle

checkpoints. These aberrant signal transduction networks can either induce or support tumor

cell development and proliferation in an organism. Cell cycle signaling through retinoblastoma

(Rb) and TGFb are two widely studied pathways upon which proliferation inducing signals

converge.

ATP

STOPTOP

10

Human A549 lung cancer cells

stained for BrdU (green) and

Ki-67 (red) using the BrdU and

Ki-67 QCI/HCA kit. Nuclei are

stained with Hoechst dye (blue).

FlowCellect Bivariate Cell Cycle kit for DnA replication (Catalogue No. FCCH025102)

Flow cytometry is an accurate, high-throughput, high content

method for studying proliferating cells undergoing DNA

replication in the S-phase of the cell cycle. The kit includes a

directly conjugated Anti-BrdU Alexa Fluor 488 conjugate plus a

DNA dye (propidium iodide) which enables the researcher to

perform bivariate analysis of proliferating cells without the

need of software modules for data interpretation.

Brdu and ki-67 QCI / HCA Assay kit (Catalogue No. HCS213)

Incorporation of BrdU into cellular DNA correlates with cell

proliferation rate and the number of cells in S phase. Ki-67

antigen is a proliferation marker preferentially expressed

during late G1, S, G2 and M phases. The BrdU and Ki-67

Quantitative Cell Imaging (QCI) kit offers an easy assay for cell

division in the presence of proliferation inhibiting factors,

either by automated microscopy or traditional (manual)

analysis. Couple this assay with other QCI kits to capture a

comprehensive picture of cell cycle progression and arrest.

MILLIPLEX map Human Src Family kinase (SFk) Multiplex Assay (Catalogue No. 48-650)

SFKs play crucial roles in embryonic development and cell

proliferation, regulating such functions as adhesion,

differentiation, and survival. These proteins are often involved

in the progression and oncogenesis of non-small cell lung

cancer (NSCLC), squamous cell head and neck and pancreatic

cancers. EMD Millipore’s MILLIPLEX map Src Family Kinase 8-Plex

is the only multiplex cell signaling kit available able to assess

and distinguish the phosphorylation status of each SFK.

Detection of DNA

replication by

analysis of S phase

cells. Bivariate flow

cytometric analysis

using BrdU Alexa Fluor

488 conjugate can

distinguish S phase cells

with great accuracy,

not only based on

their difference in DNA

content from G1 or

G2/M cells but also as

having incorporated

BrdU.

Phospho-SFK detection in cell lysates. Tumor cells (Ramos, HL-60, HeLa,

and DLD-1) were treated with pervanadate (5 minutes) to enrich for

phosphorylated proteins. Cell lysates prepared from untreated and

pervanadate-treated cells were incubated with a mixture of all eight

antibody-coated beads. Tyrosine phosphorylation of SFKs was detected

with antiphosphotyrosine antibody. Multiplex analysis (A) showed Blk, Fgr,

and Fyn phosphorylation in Ramos cells; Lck, Lyn, and Hck phosphorylation

in HL-60 cells; and Yes and Src phosphorylation in HeLa and DLD-1 cells,

respectively. The confirmatory immunoprecipitation/Western blots are

shown in (B).

Med

ian

Fluo

resc

ence

Inte

nsit

y

4,500

4,000

3,500

3,000

2,500

2,000

1,500

1,000

500

0Blk Fgr Fyn Lck Lyn Hck Yes Src

Pervanadate-treated cellsNon-stimulated cells

A.

B.

BlkIP Antibody

Pervanadate

Fgr Lck Lyn

Ramos HL-60

+- +- +- +-

IP: Specific Capture Ab Blot: Phosphotyrosine Ab

Ramos HL-60 HeLaDLD-1

FynIP Antibody

Pervanadate

Hck Yes Src

+- +- +- +-

BlkIP Antibody

Pervanadate

Fgr Lck Lyn

Ramos HL-60

+- +- +- +-

IP: Specific Capture Ab Blot: Phosphotyrosine Ab

Ramos HL-60 HeLaDLD-1

FynIP Antibody

Pervanadate

Hck Yes Src

+- +- +- +-BlkIP Antibody

Pervanadate

Fgr Lck Lyn

Ramos HL-60

+- +- +- +-

IP: Specific Capture Ab Blot: Phosphotyrosine Ab

Ramos HL-60 HeLaDLD-1

FynIP Antibody

Pervanadate

Hck Yes Src

+- +- +- +-

11

Calbiochem InhibitorSelect Protein Kinase Inhibitor Libraries

Inhibitor Characteristics• Cell-permeable†

• Potent and selective†

• ATP-competitive†

• Reversible†

• Stable in DMSO

• Structurally diverse

• Some target multiple kinases

• Known pharmacological activity

• Less toxic

Highest Quality Control• Purity by HPLC (≥95% )†

• Lot-specific data for every inhibitor in solution

• DMSO-resistant polypropylene deep-well microplates

CD-roM with comprehensive data set included in each library• Inhibitor description

• Published IC50

/Ki values

• Literature citations

• SD files

• CAS numbers

• PubChem compound ID

• Molecular weight

• Molecular structure

†Pertains to the majority of inhibitors.

tECHnoLoGY HIGHLIGHt

Interrogate multiple proliferation and survival signaling pathways simultaneously and efficiently by using InhibiorSelect

Protein Kinase Inhibitor Libraries. Targets (see comprehensive table below) include kinases important for cancer research,

such as Aurora, Bcr-Abl, Ras, Rho, and Wee1.

Target Kinases

96-well Library I

96-well Library II

96-well Library III

384-well Library I

Adenosine Kinase

X

Akt X X X

AMPK X X

ATM X X

ATR X X

Aurora X X X X

Bcr-Abl X X

CaMK X X X

Cdks X X X

cFMS X X

Chk1,2 X X X

CK1,2 X X X

c-Met X X

DAG X X X

DNA-PK X X X

eEF2EGFR X X X

ERK X X

Flt3 X X X

GSK-3 X X

IGFR X X X

IKK X X X

IP3K X

IRAK X X

JAK X X

JNK X X

Lck X X

MAPK X X X

MEK X X X

MLCK X

MNK1 X X

p70 S6 X X

P90 S6 X

PAK X

PDGFR X X

PDK1 X X

PI 3-K X X X

PIKFyve X

PIM X

PKA X

PKC X X X

PKG X

PKR X X

PLK X

Rho X X X

RIP X

SK X X

Src X X X

Syk X X

TGF-bR X X

Tpl2 X

VEGFR X X

Wee1 X

12

kEY ProDuCtS - ProLIFErAtIon CHECkPoInt

Antibodies and Proteins

Description Catalogue No.

Cell Cycle-G2/M Phase Pathway Explorer 15-120

Antibody Minipack

Anti-Wee1 06-972

Anti-Cdk5, clone DC17 05-364

Akt Magnetic Bead conjugate 16-318

Anti-Cdc42-interacting protein 4 ABS69

TGFBR-1, active 14-912

Assays

Description Catalogue No.

MILLIPLEX map Multi-species TGFb 3-Plex TGFB-64K-03

MILLIPLEX map Human Multi-Pathway Signaling Kit 48-680

FlowCellect Bivariate Cell Cycle Kit for G2/M Analysis FCCH025103

guava Cell Cycle Reagent Propidium Iodide Solution 4500-0220

Cyclin B1 and Ki-67 QCI / HCA Assay Kit HCS210

Phospho-Histone H3 (Ser-10) and Ki-67 HCS209

QCI / HCA Assay Kit

p53 and p21 QCI / HCA Assay Kit HCS235

Inhibitors

Chk2 Inhibitor II 220486*

SB 218078 559402*

Staurosporine 569397*

Cdk1 Inhibitor IV, RO-3306 217699*

*Available from EMD Chemicals Inc.

For a complete listing of cancer-related products from Millipore, please visit www.millipore.com/cancer.

For a complete listing of cancer-related products from EMD Chemicals, please visit www.emdbiosciences.com.

13

Anti-Fas (human, activating), clone CH11 (Catalogue No. 05-201)

Fas is a transmembrane receptor that activates cell death by

binding extracellular Fas ligand. Activated Fas then recruits

caspase pathway proteins to its cytoplasmic domain. This

monoclonal antibody is ideal for measuring apoptosis and

resistance to pro-apoptotic signaling by flow cytometry,

immunocytochemistry, or Western blotting. In addition, when

this antibody binds to Fas, it activates cell death in multiple cell

types. For a complete analysis, use our antibodies to

cytoplasmic and mitochondrial death signaling proteins to

measure response to pro-apoptotic (Bax, Bak, Bid, Bim) and

anti-apoptotic (Bcl-2, Bcl-xl, Bcl-W) signals.

Etoposide-, UV-, and Fas-

mediated apoptosis in Jurkat

cells. Apoptotic response,

as measured by annexin V

binding (top) and caspase

activity (bottom), was the

fastest upon stimulation

with anti-Fas antibody

(05-201). This research

was originally published in

the Journal of Biological

Chemistry. Widmann, C et

al. 1998; JBC 273(12):7141-

7147. © the American

Society for Biochemistry and

Molecular Biology.

0

0 5 10 15

25

50

75

Etoposide

UV

Fas

% A

nnex

in

posi

tive

cel

lsD

EVD

dir

ecte

d ca

spas

eac

tivi

ty (ar

bitr

ary

unit

s)

Time (hr)

0

500

1000

1500

Evading ApoptosisThe ability to elude programmed cell death (apoptosis) is a hallmark of most types of

cancer. A variety of proteins, including cell surface receptors, adaptors, proteases and

mitochondrial components, regulate a fine balance between cell survival and death by

apoptosis. Mutations of these proteins may tip the balance, resulting in the uncontrolled survival

and proliferation of cancer cells. In many cancers, aberrant regulation results in overexpression of

survivin, a potent anti-apoptotic protein. Recent studies also show that autophagy, the lysosomal

breakdown of a cell’s own components, exhibits cross-talk with apoptotic pathways, and autophagy-

deficient cells may be prone to tumorigenesis. Understanding apoptotic and autophagy signaling and

mechanisms can help exploit these pathways for therapeutic benefit.

Apoptag® ISoL Dual Fluorescence Apoptosis Detection kit (Catalogue No. APT1000)

During apoptosis, one class of DNases, which includes DNase I,

is activated via caspase signaling, while the other class, which

includes DNase II, is activated via a caspase-independent

pathway. The ApopTag ISOL Apoptosis Kit facilitates the

differentiation of apoptotic cells from necrotic or transiently

damaged cells by using a proprietary dual fluorescent-labeled

oligonucleotide to detect simultaneously both DNase I and

DNase II-type DNA fragments in a single sample, thus

distinguishing caspase-dependent and caspase–independent

apoptotic events. While conventional in situ detection

techniques such as ISEL (Klenow DNA polymerase), TUNEL

(terminal deoxynucleotidyl transferase, TdT) and ISNT (DNA

Polymerase I) are useful in detecting internucleosomal DNA

cleavage, they do not differentiate DNase Type I and DNase

Type II cleavage. The ISOL technique shows concordant results

with the TUNEL technique in specimens without necrosis, and

in specimens presenting necrosis, the ISOL technique shows

improved selectivity as compared to TUNEL.

Detection of caspase-

dependent (red) and

caspase-independent

(green) DNase activity in

paraffin-embedded rat

mammary gland tissue

sections using ApopTag

ISOL Dual Fluorescence

Apoptosis Detection Kit

(APT1000).

ATP

STOPTOP

14

Jurkat cells were uninduced (A), induced to apoptosis with staurosporine (B) or with CCCP (C), then stained using

the MitoDamage kit. Flow cytometry showed that staurosporine induced apoptosis in Jurkat cells, and that CCCP

depolarized the mitochondrial membrane, but neither condition was sufficient for cell membrane permeabilization and

death.

FlowCellect MitoDamage kit (Catalogue No.

FCCH100106)

Multiparametric, flow

cytometric evaluation of

apoptosis markers enables

detailed kinetic and temporal

analysis of the events that

lead to apoptosis, both in

individual cells and cell

populations. This kit enables

the simultaneous

measurement of 3 important

cell health parameters;

change in mitochondrial

potential (an early hallmark

of apoptosis and cellular

stress), phospatidylserine

expression on the cell

surface of apoptotic cells as

assessed by Annexin V

binding and plasma

membrane permeabilization

(indicating cell death).

MILLIPLEX map Human Apoptosis and MAPmates™ (Catalogue Nos. 48-670, 46-694, 46-692)

Apoptosis depends on coordinated regulation by multiple, interconnected pathways, therefore, it requires understanding the

roles of pathway proteins in both normal apoptosis and dysregulated cancer cells, which necessitates the quantitation of

multiple biomarkers in each sample. A portolio of MILLIPLEX map MAPmates enables the accurate, Luminex technology-based

detection of expression of key apoptosis proteins.

Single-plex Detection of Analytes Using Apoptosis MAPmates. Three different apoptosis biomarkers (active Caspase 3, phospho-histone H2A.X, and cleaved

PARP) were detected from Jurkat cells untreated or treated with 25 mM anisomycin for 4 hours using individual MAPmates. The anti-apoptotic marker phospho-

Bad was detected in A431 cells untreated or treated with 100 ng/mL EGF for 5 minutes. Insets show Western blots of active caspase 3, cleaved PARP, phospho-

H2A.X, and phospho Bad (both capture and detection antibodies). MFI represents Mean Fluorescence Intensity.

7-A

AD

Annexin V, CF488A

Red

2 F

luor

esce

nce

(RD

2-H

Log)

Green Fluorescence (GRN-HLog)

0.16%

95.2% 3.2%100 101 102 103 104

100

101

102

103

104

1.4%

Red

2 F

luor

esce

nce

(RD

2-H

Log)

Red Fluorescence (RED-HLog)

0.06%

70.5% 28.2%100 101 102 103 104

100

101

102

103

104

1.2%

Red

2 F

luor

esce

nce

(RD

2-H

Log)

Red Fluorescence (RED-HLog)

0.10%

93.9% 4.8%100 101 102 103 104

100

101

102

103

104

1.2%

Mit

oSen

se R

ed

Annexin V, CF488A

Red

2 F

luor

esec

ence

(R

D2-H

Log)

Green Fluorescence (GRN-HLog)

94.4%

0.75% 3.7%

Uninduced

100 101 102 103 104100

101

102

103

104

1.1%

Red

2 F

luor

esec

ence

(R

D2-H

Log)

Green Fluorescence (GRN-HLog)

54.7%

14.6% 27.0%

2 µM Staurosporine

100 101 102 103 104100

101

102

103

104

3.7%

Red

2 F

luor

esec

ence

(R

D2-H

Log)

Green Fluorescence (GRN-HLog)

0.20%

93.2% 6.6%

50 µM CCCP

100 101 102 103 104100

101

102

103

104

0.04%

Mit

oSen

se R

ed

Red

2 F

luor

esce

nce

(RD

2-H

Log)

Red Fluorescence (RED-HLog)

95.2%

3.2% 1.3%100 101 102 103 104

100

101

102

103

104

0.3%

Red

2 F

luor

esce

nce

(RD

2-H

Log)

Red Fluorescence (RED-HLog)

0.26%

98.4% 1.3%100 101 102 103 104

100

101

102

103

104

0.02%

7-AAD

100 101 102 103 104

101

102

103

104

Depolarized Cells

Dead Cells

Live Cells58.1% 0.08%

41.0% 0.8%

Red

2 F

luor

esce

nce

(RD

2-H

Log)

Red Fluoresecence (RED-HLog)

100

A B C

A. Active Caspase 3 B. Phospho-H2A.X C. Cleaved PArP D. Phospho-Bad

15

Cytokines

CytokineReceptor

GrowthFactors

RTKsIGF-1

IGF-1R

CaspaseCascade

GlycogenSynthesis

Translation

ProteinSynthesis

p53 DegradationNucleus

PDK1/Akt/Flt Dual Pathway Inhibitor

PDK1/Akt/Flt Dual Pathway Inhibitor,Akt Inhibitor IV, Akt Inhibitor VIII

PI-103,PI 3-Kα Inhibitor VIII

PI-103,Rapamycin

Ro-31-8220

LY 294002, PI-103,PI 3-Kγ Inhibitor,PI 3-Kγ Inhibitor II,PI 3-Kα Inhibitor IV,PI 3-Kα Inhibitor VIII,Wortmannin

ERKPathway

BCRAg

Caspase9P

PKCα

JAK1 JAK1

SYK

PDK1

AKT1mTOR

p70S6K

DNA-PK

GSK-3

Raf1

GAB1

4EBP1

eIF4E

HSP90

CDC37

BCAP

PI 3-K

PP2AMDM2

MDM2

p53

P

P

P

P

P

Calbiochem InhibitorSelect Akt/PI 3-k/mtor Signaling Pathway Inhibitor Panel (EMD Chemicals Catalogue No. 124031*)

Activation of the Akt pathway can mediate evasion of

apoptosis by tumor cells, because Akt can both inactivate

pro-apoptotic factors as well as activate transcription of

survival genes. The InhibitorSelect Akt/PI 3-K/mTOR Pathway

Panel consists of 12 highly potent and selective kinase

inhibitors and a negative control for studying the Akt

pathway in one convenient package to help elucidate

specific steps in apoptosis while staying within budget.

kEY ProDuCtS - APoPtoSIS

Antibodies and Proteins

Description Catalogue No.

Anti-Cytochrome C, clone 7H8.2C12 MAB1800

Anti-Bax, N-terminus 06-499

Anti-Bcl2, clone 100 05-729

Caspase-3, Human, Recombinant, E. coli 235417*

Assays

ApopNexin® Annexin V FITC Apoptosis Kit APT750

TUNEL Apoptosis Detection Kit 17-141

MILLIPLEX MAP Human Apoptosis – 3-Plex 48-670

FlowCellect MitoLive Kit FCCH100107

FlowCellect MitoStress Kit FCCH100109

FlowCellect Cytochrome c Kit FCCH100110

Cytochrome C QCI / HCA Assay Kit HCS236

related Inhibitor Panels

InhibitorSelect EGFR Signaling Pathway Panel 324839*

InhibitorSelect MAPK Signaling Pathway Panel 444189*

InhibitorSelect NF-kB Signaling Pathway Panel 481487*

InhibitorSelect JAK/STAT Signaling Pathway Panel 420138*

InhibitorSelect WNT Signaling Pathway Panel 681666*

Phospho-Histone H2A.X (Ser 139) and p53 QCI / kit Assay kit (Catalogue No. HCS225)

DNA damage and repair are often early indicators of

apoptosis. Phosphorylation of H2A.X is important in the

rapid formation of a stable repair complex at the site of

damage. The tumor-suppressor, p53, is involved in a complex

network, including both DNA damage and apoptosis

pathways. Together these two markers can provide insight

to the mechanism of action for how cells evade apoptosis.

Detection of DNA damage in A549 cells. A549 cells were treated for 24

hours with etoposide (left panel) or 0.4% DMSO (right). Cell handling, fixation,

and immunostaining were performed as according to HCS225 assay

protocols. Hoechst HCS nuclear stain (blue), phospho-histone H2A.X (green),

and p53 (red) fluorescence.

*Available from EMD Chemicals Inc.

For a complete listing of cancer-related products from Millipore, please visit www.millipore.com/cancer.

For a complete listing of cancer-related products from EMD Chemicals, please visit www.emdbiosciences.com.

Annotated in brown are inhibitors included in the Calbiochem InhibitorSelect

Akt/PI3K/mTOR Signaling Pathway Inhibitor Panel (124031*).

16

Family of TUNEL Assay Products for Apoptosis Detection

tECHnoLoGY HIGHLIGHt

DNA fragmentation is usually associated with

ultrastructural changes in cellular morphology in apoptosis.

The ApopTag family of kits examines apoptosis via DNA

fragmentation by the TUNEL assay. The DNA strand breaks

are detected by enzymatically labeling the free 3’-OH

termini with modified nucleotides. These new DNA ends that

are generated upon DNA fragmentation are typically

localized in morphologically identifiable nuclei and apoptotic

bodies. In contrast, normal or proliferative nuclei, which

have relatively insignificant numbers of DNA 3’-OH ends,

usually do not stain with the kit. ApopTag Kits detect

single-stranded and double-stranded breaks associated

with apoptosis. Drug-induced DNA damage is not identified

by the TUNEL assay unless it is coupled to the apoptotic

response. In addition, this technique can detect early-stage

apoptosis in systems where chromatin condensation has

begun and strand breaks are fewer, even before the

nucleus undergoes major morphological changes.

Indirect

End result of Apoptosis:Nucleosome sized DNA fragments

Step 1: Tail with digoxigenin-dNTP

Step 2: Bind antibody conjugate

Step 3: Stain withsubstrate and view bymicroscopy (peroxidase). Alternatively, analyze by microscopy or flow cytometry (fluorescein).

Direct

End result of Apoptosis:Nucleosome sized DNA fragments

Step 1: Tail with fluorescein-nucleotide

Step 2: Analyze by flow cytometry

Antibodies and Proteins

Description Catalogue No.

ApopTag Red In Situ Apoptosis Detection Kit S7165

ApopTag Fluorescein Direct In Situ Apoptosis Detection Kit S7160

ApopTag Plus Peroxidase In Situ Apoptosis Kit S7101

ApopTag Fluorescein In Situ Apoptosis Detection Kit S7110

ApopTag Plus In Situ Apoptosis Fluorescein Detection Kit S7111

ApopTag Peroxidase In Situ Apoptosis Detection Kit S7100

ApopTag Peroxidase In Situ Oligo Ligation (ISOL) Kit S7200

CaspaTag™ In Situ Apoptosis Detection Kits APT400

CaspaTag Caspase 3,7 In Situ Assay Kit, Fluorescein APT403

CaspaTag Pan-Caspase In Situ Assay Kit, Fluorescein APT420

CaspaTag Caspase 8 In Situ Assay Kit, Fluorescein APT408

CaspaTag Caspase 9 In Situ Assay Kit, Fluorescein APT409

CaspaTag Caspase 3,7 In Situ Assay Kit, Fluorescein APT423

CaspaTag Caspase 8 In Situ Assay Kit 25, Fluorescein APT428

CaspaTag Caspase 9 In Situ Assay Kit, Fluorescein APT429

CaspaTag Pan-Caspase In Situ Assay Kit, Sulforhodamine APT500

CaspaTag Pan-Caspase In Situ Assay Kit, Sulforhodamine APT520

CaspaTag Caspase 3,7 In Situ Assay Kit, Sulforhodamine APT523

17

ChIPAb+™ trimethyl-Histone H3 (Lys9) (Catalogue No. 17-625)

Telomeres are normally enriched in epigenetic marks that are

characteristic of heterochromatin, such as di- and

trimethylated H3K9 (H3K9me3) and H4K20. These marks, along

with phosphorylation of H3 (Ser10) and others, are correlated

with chromatin condensation and active replication. In

contrast, epigenetic marks such as phospho-H2A.X (Ser139)

and phospho-H2B (Ser14) mark active apoptosis and DNA

damage. The ChIPAb+ validated antibody/primer set for

chromatin immunoprecipitation of H3K9me3 complements EMD

Millipore’s broad range of specific, highly validated antibodies

and assays for epigenetic signaling pathways.

CpG MethylQuest™ DnA Isolation kit (Catalogue No. 17-10035)

Aberrant hypermethylation of normally unmethylated CpG

islands is a frequent event in immortalized and transformed

cells, and is associated with transcriptional inactivation of

tumor suppressor genes. DNA methylation also correlates with

repressed telomere recombination. EMD Millipore’s CpG

MethylQuest DNA Isolation Kit provides an efficient, convenient

solution for mapping methylation across a single gene or

across the entire genome, using a unique GST-methyl-CpG

binding domain fusion protein that tightly binds CpG

methylated sequences but has extremely low affinity for

non-methylated regions.

Sonicated chromatin prepared from 3x106 NIH 3T3 L1 cells was subjected

to chromatin immunoprecipitation using 4 µg of either normal rabbit IgG

or Anti-trimethyl-Histone H3 (Lys9) antibody (17-625) and the Magna ChIP

A kit (17-610). Successful enrichment of trimethyl-histone H3 (Lys9)-

associated DNA fragments was verified by qPCR using included ChIP

primers flanking the mouse p16 promoter.

HeLa and MCF7 genomic DNA were purified with the CpG MethylQuest. DNA

isolation kit. 2 µL of the purified samples were used for 30 cycles of PCR

amplification of SNRPN (frequently methylated locus) and GAPDH (typically

unmethylated). PCR aliquots were loaded on agarose gels (S = supernatant,

not bound to beads; E = elution, bound to beads)

0

10

20

30

40

H3K9me3IgG

Fold

Enr

ichm

ent

1.00

34.93

Limitless Replicative PotentialIn normal cells, telomeres protect chromosomes from fusing with each other or

rearranging during mitosis. Telomeres become shorter with each cell division, limiting

cells to a fixed number of divisions. Tumor cells can achieve unlimited replicative potential

either by synthesizing high levels of telomerase enzyme or via homologous recombination to create

lengthened telomeres. Upregulation of telomere maintenance occurs in about 90% of human

cancers. Increasing evidence indicates that chromatin modifications are important regulators of

telomeres. Loss of epigenetic regulation correlates with aberrant telomere length. These links

between epigenetic status and telomere-length regulation provide important insights for analyzing

cancer progression and aging.

HeLa S HeLa E MCF7 S

Positive: SNRPN Negative: GAPDH

MCF7 E HeLa S HeLa E MCF7 S MCF7 E

HeLa S HeLa E MCF7 S MCF7 E

Positive: SNRPN

Negative: GAPDH

ATP

STOPTOP

18

Fluorescent Conjugated Antibodies for Cell Surface CD Markers (Catalogue Nos. FCMAB211F, FCMAB188F and others)

The replicative potential of cancer cells is analogous to that

of stem cells. In fact, many studies have shown tumors’

stem-like characteristics to correlate with malignancy. One

strategy to isolate cancer cells with high replicative

potential is using flow cytometry to identify cells that

express stem cell surface markers, such as CD90 and CD34.

EMD Millipore’s growing selection of directly conjugated

antibodies for cancer-relevant CD markers is specifically

designed, optimized, and validated for flow cytometry.

Phospho-Histone H3(Ser10) and Cyclin B1 QCI /HCA Assay kit (Catalogue No. HCS211)

Cyclin B1 and histone H3, active in chromatin condensation,

play key roles in DNA replication and mitosis. QCI of histone

H3 phosphorylation and cyclin B1 expression have proven to

be powerful tools in drug discovery; for example, High

Content Analysis (HCA) of histone H3 phosphorylation has

been used to characterize Aurora kinase inhibitors. HCA of

cyclin B1 expression is used for cell cycle inhibitor profiling.

Calbiochem Brdu Cell Proliferation Assay (EMD Chemicals Catalogue No. QIA58*)

DNA replication in normal and tumor cells can be accurately

measured by assessing the degree to which BrdU, a modified

nucleotide, is incorporated into DNA. This BrdU Cell

Proliferation Assay is a non-isotopic immunoassay for

quantification of BrdU incorporation into newly synthesized

DNA of actively replicating genomes. It is sensitive, rapid,

and easy to perform.

Human peripheral blood

lymphocytes were

stained with Milli-Mark™

human CD90-FITC

antibody and analyzed

by flow cytometry. The

right-hand (lower) peak

represents CD90-

expressing cells.Immunofluorescence of untreated and cell cycle-arrested HeLa cells. HeLa

cells were treated with either 0.4% DMSO control (left) or the G2/M phase

cell cycle arresting agents, vinblastine sulfate (top right) or paclitaxel

(bottom right). The cells were fixed and stained for Phospho-Histone

H3(Ser10) (top, green) or Cyclin B1 (bottom, red) and Hoechst nuclear stain

(blue). The percentage of cells expressing phospho-histone H3 or nuclear

cyclin B1 increased following arrest of cells in M phase.

The Calbiochem BrdU Cell Proliferation Assay responds to increasing cell

number with higher sensitivity than a competing assay.

00.2 0.5 1.0 4.0

19 hr BrdU Calbiochem Assay

2 hr BrdU Calbiochem Assay

19 hr BrdU Competitor Assay

2 hr BrdU Competitor Assay

10.0 100.0

Cell Number/Well x 10

Sensitivity of Calbiochem BrdU Cell Proliferation Assayvs. Competitor BrdU Labeling Kit

40

80

120

S/N

(si

gnal

/sig

nal

of n

o ce

lls)

kEY ProDuCtS - rEPLICAtIvE PotEntIAL

Antibodies and Proteins

Description Catalogue No.

Anti-phospho-Histone H2A.X (Ser139), clone JBW301 05-636

Anti-phospho-Histone H2B (Ser14) 07-191

Anti-HDAC9, clone LH/JC2 05-897

Aurora A, active purified kinase 14-511

Anti-CD34 Class III, clone 581, FITC conjugated CBL555F

Assays

CpGenome Turbo Bisulfite Modification Kit S7847

CpG WIZ® ERa Amplification Kit S7815

TRAPeze® Telomerase Detection Kit S7700

BrdU Cell Proliferation Assay, HTS HTS01*

BrdU Immunohistochemistry System HCS30*

Senescence Detection Kit QIA117*

p53 and p21 QCI / HCA Assay Kit HCS235

*Available from EMD Chemicals Inc.

For a complete listing of cancer-related products from

Millipore, please visit www.millipore.com/cancer.

For a complete listing of cancer-related products from EMD Chemicals, please visit www.emdbiosciences.com.

19

Anti-vEGF, clone JH (Catalogue No. 05-443)

Vascular endothelial growth factor (VEGF) is a pro-angiogenic

growth factor both secreted by tumor cells and expressed in

response to hypoxia via HIF signaling. Specially validated for

immunohistochemistry of paraffin-embedded tissues, this

antibody complements EMD Millipore’s comprehensive

offering of antibodies against targets of various stimulating

mechanisms of angiogenesis, including various VEGF

isoforms, VEGF Receptors, FGFs, and angiopoietins.

In Vitro vascular Permeability Assays (24- & 96-Well) (Catalogue Nos. ECM644, ECM642)

The In Vitro Vascular Permeability Assay provides an efficient

system for evaluating the effects of chemicals and drugs on

endothelial cell adsorption, transport, and permeability.

Endothelial cells are seeded onto collagen-coated inserts in

multiwell plates and treated with cytokines, growth factors,

or any reagent of interest. After treatment, the extent of

permeability is determined by measuring the fluorescence of

the plate well solution.

Immunohistochemistry of paraffin-embedded human breast carcinoma

stained with anti-VEGF (05-443).

Permeability analysis of HUVEC monolayers. HUVEC cells were cultured, treated

with TNFa, then subjected to FITC-dextran permeability testing and monolayer

staining. Data showed high permeability in the absence of an occlusive

endothelial cell monolayer. The “No Treatment” sample exhibits a visually

confluent monolayer, as supported by low FITC-dextran permeability. Disruption

of monolayer integrity is observable both visually and by quantification of

increased plate well solution fluorescence following TNFa treatment.

Sustained AngiogenesisAngiogenesis, the development of new vascular networks, is rare in adult tissues. In

tumors, dysregulated signaling and hypoxic conditions lead to sustained, almost

uncontrolled angiogenesis, a necessary component of tumor growth and metastasis. Chronic

inflammation mechanisms, such as the production of reactive oxygen species during infection and

secretion of pro-inflammatory cytokines, can also foster angiogenesis in tumor progression.

Angiogenic signaling in tumors is similar to normal angiogenesis, mediated by soluble growth factors,

membrane-bound receptors, and cell-cell and cell-matrix interactions. Such signaling regulates cell

migration, which is vital to angiogenesis. However, there are multiple differences between tumor

angiogenesis and normal blood vessel formation. Tumor endothelial cells proliferate faster than

non-tumor endothelial cells. Tumor vasculature differs from normal vasculature in morphology,

enhanced leakiness, and structural abnormalities. Finally, tumor vessels are often not capable of

transporting oxygen to and removing waste products from all of the tumor tissues, resulting in

frequent tumor cell necrosis.

no Monolayer

100 ng/mL tnF-a

no treatment

NoMonolayer

NoTreatment

100 ng/mL TNF-α

30

20

10

Fluo

resc

ent

Cou

nt

( x 1

00

0)

0

NoTreatment

100 ng/mL TNF-α

2.0

1.0

0.5

1.5

Fluo

resc

ent

Cou

nt

( x 1

00

0)

0

NoMonolayer

NoTreatment

100 ng/mL TNF-α

30

20

10

Fluo

resc

ent

Cou

nt

( x 1

00

0)

0

NoTreatment

100 ng/mL TNF-α

2.0

1.0

0.5

1.5

Fluo

resc

ent

Cou

nt

( x 1

00

0)

0

ATP

STOPTOP

20

MILLIPLEX map Human Cytokine/Chemokine Panel I(Catalogue No. HCYTOMAG-60K)

Response to anti-angiogenic chemotherapy is highly dependent on the genetic profile of both the individual and the tumor. For

example, preclinical studies of the vascular targeting cancer drug candidate NSC 640488 demonstrated two processes critical

for anti-tumor effects: (1) tumor vascular endothelial apoptosis and (2) production of anti-vascular and pro-immunity

cytokines1,2. This MILLIPLEX map multiplex analyte panel enables characterization of multiple biomarkers associated with

individual genetic profiles simultaneously, increasing the sensitivity and specificity of the assay while conserving precious

samples.

References1. Jassar AS et al. Activation of tumor-associated macrophages by the vascular disrupting agent 5,6-dimethylxanthenone-4-acetic acid induces an effective

CD8+ T-cell-mediated antitumor immune response in murine models of lung cancer and mesothelioma. Cancer Res. 2005 Dec 15;65(24):11752-61.

2. Ching LM et al. Induction of endothelial cell apoptosis by the antivascular agent 5,6-Dimethylxanthenone-4-acetic acid. Br J Cancer. 2002 Jun

17;86(12):1937-42.

In a clinical trial for the anticancer drug NSC 640488, researchers used the MILLIPLEX map Human Cytokine 42-plex assay to identify a consistent pattern of

cytokine/chemokine modulation in the peripheral blood lymphocytes (PBL) of human donors that responded best to NSC 640488. Shown are fold changes in

cytokine concentrations in PBLs from subjects treated with the drug as compared to untreated PBLs from different donors. A small cohort of donors showed

downregulation of IP-10, MCP-1, and sCD40L and increased induction of TNFa, MIP-1a, IL-6, and IL-8. This pattern of detection may potentially correlate with a

patient’s responsiveness to NSC 640488: high responders (group A) or low responders (group B). NC denotes less than a 1.5-fold change.

Group Aα α α

ααα

21

ready-to-Assay™ CXCr2 Chemokine receptor Frozen Cells (Catalogue No. HTS002RTA)

The chemokine IL-8 is highly expressed in many cancers; IL-8

behaves as a chemoattractant for endothelial cells

expressing IL-8-interacting CXCR2 receptors, promoting

their migration to cancers, ultimately facilitating

angiogenesis. Ready-to-Assay CXCR2 frozen cells are fully

validated for assessing the functional activity of CXCR2-

interacting compounds, providing cell-based assay data

while eliminating time-consuming cell culture.

Calcium flux in CXCR2–expressing Chem-1 cell line induced by rhGROα.

CXCR2–expressing Chem-1 Ready-to-Assay cells were loaded with a calcium

dye, and calcium flux in response to the indicated ligand (4-fold serial

dilution with each concentration performed in duplicate) was determined on

a Molecular Devices FLIPRTETRA®. Maximal fluorescence signal obtained in

this experiment was 2,500 RLU (Relative Light Units).

0

-12 -11 -10 -9 -8 -7

rhGROα

-6

20

40

60

80

100

120

Rel

ativ

e Li

ght

Uni

ts(%

of

max

imum

)

Log [Dopamine], M

kEY ProDuCtS - AnGIoGEnESIS

Antibodies and Proteins

Description Catalogue No.

Anti-Angiopoietin-1 AB10516

Anti-VEGF Receptor-3, extracellular domain, MAB3757

clone 9D9F9

Anti-FGF-2/basic FGF (neutralizing), clone bFM-1 05-117

Flt-1(VEGFR1) active purified kinase 14-562

Assays

In Vitro Angiogenesis Assay Kit ECM625

Fibrin In Vitro Angiogenesis Assay ECM630

MILLIPLEX map Human Cytokine/Chemokine HCYP2MAG-62K

Magnetic Bead Panel II

MILLIPLEX map Human Soluble Cytokine Panel HSCR-32K

ChemiScreen™ CXCR2 Chemokine Receptor HTS002M

Membrane Preps

Ready-to-Assay EP2 Prostanoid Receptor HTS185RTA

Frozen Cells

related Inhibitors

InhibitorSelect VEGF Signaling Pathway Panel 676502*

TAS-301 608050*

VEGF Inhibitor, CBO-P11 676496*

Withaferin A, Withania somnifera 681535*

*Available from EMD Chemicals Inc.

For a complete listing of cancer-related products from

Millipore, please visit www.millipore.com/cancer.

For a complete listing of cancer-related products from EMD Chemicals, please visit www.emdbiosciences.com.

22

Millicell® µ-Angiogenesis Assay Kits

tECHnoLoGY HIGHLIGHt

(Catalogue Nos. MMA125 and MMA130)

Quantify activation or inhibition of tumor angiogenesis with Millicell µ-Angiogenesis Assay Kits. These kits provide a

powerful, quantitative, slide-based platform for superior, real-time visualization of tubule formation in angiogenesis. The kits

are optimized and easy to use with properties that prevent meniscus formation and out-of-focus areas. An entire well can

be visualized at low magnification, generating enormous time and cost savings.

Millicell µ-Angiogenesis Activation Assay. The angiogenesis activation kit includes fibrinogen and thrombin components to make the fibrin gel, the activation

control PMA (phorbol myristate acetate), and calcein-AM to visualize the cells in real time. (Left to right and top to bottom) Bright field, calcein-AM, DAPI, and

calcein-DAPI merge micrographs show robust tubule formation and lumen structure in the presence of phorbol myristate acetate (PMA, a proangiogenic agent).

Millicell µ-Slide vs. 96-Well Plate

no meniscus• Improved optics

• All cells in focus

• 10 µL inner well volume

• 15 wells

• Slide format

10x 10x

DepthFocus

unique well design eliminates meniscus• Upper well contains

media/reagents

• Inner well contains

cells and/or matrix

Meniscus formation• Conventional optics

• Few cells in focus

• 100 µL well volume

• 96 wells

• Multiwell format

With no stimulant Lumen StructureWith 50 µg/mL PMA and 1 x ITS

Grow, treat, stain, and visualize cells all in one device:

Enhanced imaging capabilities of Millicell slides improve studies of angiogenesis:

23

Anti-Hypoxia Inducible Factor 1a (HIF-1a)(Catalogue No. 07-628)

Tumor-specific altered metabolism (the “Warburg

Phenomenon”) consists of an increase in aerobic glycolysis.

A principal mechanism of aerobic glycolysis involves

activation of HIF-1a, overexpressed in many cancers. HIF-1a

also promotes increased vascularization and tumor

progression. This antibody is validated for

immunoprecipitation, immunohistochemistry, and Western

blotting, and complements EMD Millipore’s comprehensive

offering of antibodies related to tumor-specific alterations

of celluar metabolism, including antibodies for receptor

tyrosine kinases, the PI3K-AKT signaling pathway, and

mitochondrial detection.

Calbiochem Anti-PDH-E1a (pSer293) rabbit pAb (EMD Chemicals Catalogue No. AP1062*)

Dysregulation of the pyruvate dehydrogenase (PDH) complex

(PDC) in skeletal muscle has been implicated in type 2

diabetes and various mitochondrial diseases. Recently, it has

been shown that inhibition of PDC activity in cancer cells

promotes Warburg metabolic and malignant phenotype. The

activity of PDC is mainly regulated by the phosphorylation

state of Ser293, Ser232, and Ser300 on the E1a subunit.

Modified nuclear extract

from normal MCF-7

cells (Lane 1) and MCF-7

cells treated with 150

mM cobalt chloride (a

hypoxia mimetic) for

16 hours (Lane 2) and

analyzed by Western

blotting with anti-HIF-

1a (07-628).

Detection of phospho-PDH-E1a (Ser293) by immunocytochemistry. COS7

cells were incubated in dichloroacetate (DCA), an inhibitor of PDH kinase.

All samples were incubated with mitochondrial stain (red), fixed and

permeabilized. Primary antibodies: PhosphoDetect™ Anti-PDH-E1a (pSer293)

Rabbit pAb (Cat. No. AP1062, green, top) or anti-PDH-E1a antibody (green,

bottom). Detection: fluorescence (Alexa Fluor 488 secondary antibody) with

DAPI (blue). Data courtesy of Sandra Wiley and Matthew Rardin, University

of California, San Diego.

116

66

97

45

1 2

2 hrDCA

2 hrDCA

Mitochondria MergePhospho-

PDH

Metabolic ReprogrammingNormal cellular metabolism involves a complex series of controlled biochemical

reactions that produce energy in the form of ATP to maintain homeostasis and allow

cells to respond to environmental changes. These biochemical reactions are disrupted during

tumorigenesis as cancer cell proliferation results in increased distances from vascular basement

membranes, causing regional hypoxia. When ATP production from glucose metabolism falls below

maintenance levels, there is an upregulation of anaerobic glycolysis that can then become

permanent through the stabilization of hypoxia-inducible factor 1a (HIF-1a) and/or the upregulation

of phosphorylated c-myc. This glycolytic adapatation causes regional acidosis, which provides

growth advantages to the cancer population, allowing tumor cells to modify their environment in a

way that is toxic to non-tumor cells. The acidic environment also leads to increased motility and

cancer cell invasion into adjacent normal tissue, putting cancer cells in direct contact with

mesenchymal cells.

ATP

STOPTOP

24

A. U937 cells were stimulated with IFNg, IL-6, and GMCSF, then stained with antibodies against phospho-STAT1, phospho-STAT3, and phospho-STAT5A/B. Double

positive cells (blue) are seen in each plot, indicating simultaneous detection of all three activated STAT proteins. Untreated U937 cells (red) are shown for

comparison. B. Single parameter overlays of activated STAT proteins in untreated (red) vs. treated (blue) U937 cells stimulated with IFNg for STAT1 activation, IL-6

for STAT3 activation, and GM-CSF for STAT5A/B activation.

HIF-1a EZ-tFA transcription Factor Assays (Catalogue Nos. 70-670, 70-570, 70-500)

The transcription factor HIF1a is a key regulator of the

hypoxia response, mediating both metabolic reprogramming

of tumor cells as well as stimulating angiogenesis. EMD

Millipore’s HIF-1a EZ-TFA Transcription Factor Assays are

powerful tools for measuring the DNA binding activity of

HIF-1a in nuclear extracts. The assays are provided in

non-radioactive, 96-well formats.

FlowCellect Multi-StAt Activation Profiling kit (Catalogue No. FCCS025550)

STAT transcription factors regulate cell proliferation, survival, and migration downstream of transmembrane receptors.

Recent research reports additional roles for STATs in metabolic reprogramming. Active, nuclear STAT3 increases transcription

of HIF-1a, promoting tumorigenesis, while mitochondrial STAT3 helps mediate transformation by affecting glycolysis and

oxidative phosphorylation. By providing simultaneous detection of phosphorylated STATs, the FlowCellect Multi-STAT

Activation Kit enables quick profiling of the status of constitutive activation of STAT1, STAT3, and STAT5 within a population

of cells or tumors.

Nuclear extract from CoCl2-treated Cos-7 cells were assayed with the EZ-

TFA HIF-1a assay kit and showed robust binding to the DNA capture probe,

which is complementary to the endogenous HIF-1a DNA-binding sequence,

the hypoxia response element (HRE). Specific binding was confirmed, as

addition of specific competitor oligonucleotide reduced the observed

signal. Signal was further diminished in the absence of DNA capture probe

(“Negative Control”).

0CoCl2 +

Cos-7 cellsCompetitor Negative

ControlBackground

cps

( x

10

6)

8

10

12

6

4

2

104

103

102

101

100

100 101 102 103 104

104

103

102

101

100

100 101 102 103 104

104

103

102

101

100

100 101 102 103 104

pSTAT3-Alexa® 488 (GRN-HLog) pSTAT5-PE (YLW-HLog) pSTAT5-PE (YLW-HLog)

STAT1-PerCP (RED-HLog) STAT3-Alexa 488 (GRN-HLog) STAT5-PE (YLW-HLog)

100

0

48

95

143

190

101 102 103 104

Cou

nt

pSTA

T1-P

erC

P

pSTA

T1-P

erC

P

pSTA

T3-A

lexa

488

100

0

63

125

188

250

101 102 103 104

Cou

nt

100

0

53

105

158

210

101 102 103 104

Cou

nt

A.

B.

25

MILLIPLEX map Akt/mtor Multiplex Assay (Catalogue No. 48-611)

The Akt/mTOR pathway regulates production of key

metabolic effectors:

• RNA Polymerase I and III: protein synthesis, cell

metabolism

• HIF-1a and b: angiogenesis, cell metabolism, proliferation,

motility

Akt signaling is initiated by binding of growth factors to

receptors such as the insulin receptor and EGFR.

Dysregulation of Akt/mTOR pathways results in multiple

disease states, including cancer. The MILLIPLEX map Akt/

mTOR panel uses the Luminex xMAP-based platform to

detect simultaneously changes in 11 pathway targets.

kEY ProDuCtS - MEtABoLISM

Antibodies

Description Catalogue No.

Anti-Hypoxia Inducible Factor 1a (HIF-1a), MAB5382

clone H1a67

Anti-Mitochondria, surface of intact mitochondria, MAB1273

clone 113-1

Anti-PI3 Kinase, p110a 09-481

Anti-PDH-E1a (pSer232) Rabbit pAb AP1063*

Anti-PDH-E1a (pSer300) Rabbit pAb AP1064*

Anti-BRCA1 (Ab-1) Mouse mAb (MS110) OP92*

Assays

Description Catalogue No.

Mitochondria/Cytosol Fractionation Kit MIT1000

MILLIPLEX map Akt/mTOR – 8 Plex 48-611

MILLIPLEX map Total HIF-1a MAPmate 46-665

MILLIPLEX map GSK3b (Ser9) MAPmate 46-690

FlowCellect EGFR/STAT1 Activation FCCS025142

Dual Detection Kit

FlowCellect EGFR/STAT3 Activation FCCS025143

Dual Detection Kit

Effects of inhibition and stimulation of HepG2 cells on Akt/mTOR phospho-

proteins. To demonstrate the power of the Akt/mTOR panel, HepG2 cells

were treated with specific inhibitors such as wortmannin, which blocked

phosphorylation of mTOR, TSC2, Akt, GSK3a, GSK3b, p70S6K, and RPS6.

This was followed by stimulation with 50 ng/mL IGF1 or 1 µM insulin for 15

minutes. Various phosphorylated biomarkers were simultaneously detected

by the Luminex xMAP platform (represented as average MFI) using EMD

Millipore’s Akt/mTOR panel.

2,000

p70S6

KIRS

GSK3α

IGF1R

GSK3β Akt

PTEN IR

RPS6

TSC2

mTOR0

MFI

4,000

6,000

HepG2 + InhibitorsHepG2 + Insulin

*Available from EMD Chemicals Inc.

For a complete listing of cancer-related products from Millipore, please visit www.millipore.com/cancer.

For a complete listing of cancer-related products from EMD Chemicals, please visit www.emdbiosciences.com.

26

Anti-FoXP3, clone 3G3 (Catalogue No. 04-960)

FOXP3 regulates the development and differentiation of T

regulatory T-cells in the normal immune system. However,

many cancers express high levels of FOXP3, suggesting that

it mediates tumor escape from immune control. This FOXP3

antibody is validated for flow cytometry and ELISA, making it

ideal for multiparametric studies of the immune system. It

complements EMD Millipore’s wide range of antibodies for

studying immune cell regulation including antibodies against

IL-17, CTLA4, various cytokines and chemokines, and

members of the JAK/STAT pathway.

FlowCellect Human FoXP3 treg Characterization kit (Catalogue No. FCIM025118)

Regulatory T-cells suppress the activation of the immune

system following infections, maintaining homeostasis. Some

cancer patients have increased numbers of regulatory

T-cells, allowing malignant cells to escape the activation of

the immune system. This kit helps phenotypically distinguish

and quantify human regulatory T-cells with high accuracy

and specificity. Our combination of directly conjugated

antibodies and optimized buffers enable simultaneous

detection of CD3 and CD4 on the surface and FOXP3 in the

nucleus.

FoxP3 is expressed in

mouse splenocytes.

Surface staining of

mouse CD4+ CD25+

splenocytes with 5 µg

of a biotinylated version

of Anti-FOXP3, clone

3G3 (open histogram) or

with mouse biotinylated

IgG isotype control

(shaded histogram).

Cells were detected

using Streptavidin-FITC

and analyzed using flow

cytometry. Human PBMCs were stimulated with IL-2 for 2 days. Total T-cells (CD3-

positive lymphocytes) were analyzed for dual expression of CD4 and FOXP3

(shown in the upper right quadrant of right plot). 3.25% of the T-cells for

this sample are CD3+, CD4+, and FOXP3+. A CD3 gate is used to eliminate

cell types other than T-cells, such as monocytes, which can also express

FOXP3, to avoid false positives.

32

0100 101 102

FL1-H

103 104

Escape from Immune ControlUnlike other harmful agents that invade the body, cancer cells avoid immune control by two

major mechanisms: first, they avoid antigenic recognition by immune cells, and second, they

neutralize immunogenic response. Cancer cells avoid antigenic recognition by hiding within difficult-

to-access tissues and by shedding adhesion molecules and antigens that would otherwise attract

immune cells. Cancer cells neutralize the immunogenic response by secreting immunosuppressive

cytokines (such as TGFb, IL-10, and VEGF) and by secreting anti-apoptotic signals. Cancer-driven

suppression of immune response can potentially impact the way in which the host immune system

recruits immune regulatory cells, uses inhibitory receptor-ligands, and produces inhibitory or

inflammatory cytokines. Resulting dysfunctions in the host’s own immune system further facilitate

evasion of immune control by cancer cells.

Anti-CD3-PE/Cy5 Anti-CD4-FITC

ATP

STOPTOP

Ant

i-FO

XP3-A

lexa

Flu

or 6

47

Sid

e Sca

tter

(SSC)

27

MILLIPLEX map Human Circulating Cancer Biomarker Panel 1 (Catalogue No. HCCBP1MAG-58K, magnetic)

Understanding the role of cytokines and chemokines

secreted either by tumors to avoid immune control or by the

body to effect immune response or inflammation has shed

light on the interactions between a tumor and its

environment. This multiplexed analyte panel consists of 26

circulating cancer biomarkers that include cytokines,

chemokines, and metabolic markers. Some biomarkers are

tumor type-specific, such as prostate-specific antigen (PSA),

while others, such as IL-8, have been detected in many

cancers.

Detection of circulating cancer biomarkers using the MILLIPLEX map Human

Circulating Cancer Biomarker Panel 1 in normal, lung and prostate cancer

samples.

kEY ProDuCtS - IMMunE ControL

Antibodies

Description Catalogue No.

Anti-CTLA4 (CD152), clone 9H10 04-963

Anti-JAK3, clone HL423 04-011

Assays MILLIPLEX map Human Sepsis Panel 1 HSEP-63K

FlowCellect Mouse FoxP3 Identification Kit FCIM025126

Flowcellect Human Lymphocyte ZAP-70 FCIM025122

Characterization Kit

FlowCellect Mouse Th1/Th17 FCIM025138

Intracellular Cytokine Kit

Inhibitors

17-AAG 100068*

Geldanamycin, Streptomyces hygroscopicus 345805*

Heat Shock Protein Inhibitor I 373260*

Hsp25 Kinase Inhibitor 385880*

Con

cent

rati

on

4

3

2

1

0

200

150

100

50

Normal

CYFR

A21-1

FGF2 IL6

IL8

Prl

Lept

inCA

125

sFas

OPN

sFas

lTN

F-α

MIF

AFP

HG

FH

CG

-βH

E4CA

19-9

CA

15-3

VEG

FSCF

TR

AIL

TG

F-α

Toal

PSA

CEA

Con

cent

rati

on

30

25

20

15

10

5

4

3

2

1

0

Lung Cancer

CYF

RA

21-1

FGF2 IL6

IL8

Prl

Lept

inCA

125

sFas

OPN

sFas

lTN

F-α

MIF

AFP

HG

FH

CG

-βH

E4CA

19-9

CA

15-3

VEG

FSCF

TR

AIL

TG

F-α

Toal

PSA

CEA

Con

cent

rati

on

15000

10000

5000

0

Prostate Cancer

CYF

RA

21-1

FGF2 IL6

IL8

Prl

Lept

inCA

125

sFas

OPN

sFas

lTN

F-α

MIF

AFP

HG

FH

CG

-βH

E4CA

19-9

CA

15-3

VEG

FSCF

TR

AIL

TG

F-α

Toal

PSA

CEA

*Available from EMD Chemicals Inc.

For a complete listing of cancer-related products from

Millipore, please visit www.millipore.com/cancer.

For a complete listing of cancer-related products from EMD Chemicals, please visit www.emdbiosciences.com.

28

Typical phospho-Met

(Tyr1230/Tyr1234/Tyr1235)

standard curve. 100 µL of

progressive 2 fold dilutions

of the Met standard was

analyzed as described in the

assay instructions.

Anti-Fibronectin, CS-1 segment, clone P1F11 (Catalogue No. MAB1939)

Fibronectin is a surface glycoprotein and one of many

adhesion molecules used by tissue cells to interact with and

invade the host tissue. Fibronectin can bind to collagen,

fibrin, heparin, and actin. This monoclonal antibody, validated

for multiple applications, complements EMD Millipore’s broad

portfolio of antibodies for probing the metastatic potential

of tumor cells and studying mechanisms of tissue invasion,

including antibodies to actin, integrins, MMPs, cadherins,

selectins, and other extracellular matrix (ECM) proteins and

cell adhesion molecules.

Phospho-Met (tyr1230/tyr1234/tyr1235) StAr ELISA kit (Catalogue No. 17-470)

Mutations in the Met tyrosine kinase are common in many

cancers, and Met controls cell migration and metastasis.

This colorimetric STAR (Signal Transduction Assay Reaction)

ELISA kit is a solid phase sandwich enzyme linked

immunosorbent assay that provides a fast, sensitive method

to detect specific levels of phosphorylated Met in whole cell

extracts.

Fibronectin in normal mouse gut mucosa detected by immunohistochemistry

with anti-fibronectin, CS-1 (MAB1939).

1.25

Abs

orba

nce

at 4

40

nM

Typical phospho-Met (Tyr1230/Tyr1234/Tyr1235)

Unit/mL

0 50

1.00

0.75

0.50

0.25

0.00100

Tissue Invasion and MetastasisMetastasis is the cumulative result of multiple changes in tumor cells and their

microenvironment that enable cells to migrate to locations distant from the primary tumor

and colonize the host tissue. Rampant angiogenesis facilitates metastasis, providing nutrients to

the periphery of the primary tumor and easily penetrable vascular walls. Invasion of the stroma and

vessel walls requires tumor cells to downregulate cell-cell adhesion (by altering expression of

surface molecules and mimicking epithelial-mesenchymal transition (EMT)) and secrete matrix-

degrading enzymes. After tumor cells survive circulation, they adhere to endothelial cells at the

metastatic site, extravasate, and colonize the new host tissue. The ability to colonize distant sites

is mediated both by tumor cell adhesion as well as premetastatic changes in the distant

microenvironment that prepare for arriving metastatic cells. The mechanisms by which this

“metastatic niche” is determined are still being studied.

ATP

STOPTOP

29

QCM™ Cell Invasion/Migration/Chemotaxis Assays (Catalogue Nos. ECM551, ECM558, ECM510, ECM580

and others)

The most widely accepted in vitro approach to evaluating cell

invasion as a function of metastasis is the Boyden chamber

method. The porous membrane insert of a multiwell plate is

layered with an extracellular matrix (ECM) or basement

membrane protein to simulate the barriers invaded by tumor

cells in vivo. EMD Millipore’s QCM inclusive invasion, migration,

and chemotaxis assays enable the investigation of various

elements of cell movement during metastasis.

ChemiScreen CXCr4 Chemokine receptor Membrane Preparations (Catalogue No. HTS004M)

Many cancers overexpress CXCR4 chemokine receptors,

which likely facilitate their invasive and metastatic potential.

Various tissues and organs release SDF1, a chemoattractant

and the endogenous ligand for CXCR4 receptors. CXCR4

receptors likely contribute to metastasis by guiding cells

towards tissues enriched with SDF1. EMD Millipore’s CXCR4

membrane preps enable accurate, efficient identification of

molecules that can compete for SDF1 binding to CXCR

receptors.

MILLIPLEX map Human MMP Panels 1 and 2 (Catalogue Nos. HMMP1-55K, HMMP2-55K)

The MMPs and tissue inhibitors of metalloproteases (TIMPs)

are responsible for the balance in the breakdown of

extracellular matrix (ECM), playing a key role in normal

physiological processes, such as embryonic development and

tissue morphogenesis. Disruption of the MMP/TIMP balance

can result tumor growth and metastasis. The MILLIPLEX map

Human MMP and TIMP Panels enable you to explore the

modulation of TIMP expression and the function of the MMP/

TIMP balance.

Matrix metalloprotease (MMP) inhibitor blocks invasion by HT-1080 cells.

After applying a general MMP inhibitor (GM6001) to both a metastatic

cancer cell line (HT-1080) and normal fibroblast cell line (NIH 3T3), cell

invasion was initiated using 10% FBS as a chemoattractant and measured

using the QCM Cell Invasion Assay (ECM551).

Competition binding for

CXCR4. 5 mg/well CXCR4

Membrane Preparation

was incubated with

125I-labeled SDF-1a

(0.065, 1.3 or 0.25

nM), and increasing

concentrations of

unlabeled SDF-1a, and

4-fold signal:background

was obtained.

25

[125 I

]-S

DF-

1α b

ound

(cpm

x 1

,00

0)

[unlabeled SDF-1α] log M

-14 -13

20

15

10

5

0-12 -11 -10 -9 -8

0.065 nM SDF-1α

0.13 nM SDF-1α

0.25 nM SDF-1α

-7 -6 -5

kEY ProDuCtS

Metastasis Antibodies and Proteins

Description Catalogue No.

Anti-Integrin aVb3, clone LM609, azide free MAB1976Z

Anti-MMP-9, catalytic domain AB19016

Anti-Met (extracellular), clone 4F8.2 05-1049

Met (D1246N), active 14-818

TIMP-2, human, recombinant PF021*

Metastasis Assays Endothelial Cell Adhesion Assay ECM645

Integrin-mediated & ECM530 and others

ECM Cell Adhesion Arrays

Quantitative Pseudopodia Assay Kit ECM650

MILLIPLEX map Human TIMP Panel 2 HTIMP2-54K

Ready-to-Assay LPA1 Lysophospholipid HTS089RTA

Receptor Frozen Cells

ChemiScreen EP2 Prostanoid Receptor Membrane Preps HTS185M

ChemiScreen LPA1 Lysophospholipid Membrane Preps HTS089M

Metastasis Inhibitors GM6001 (MMP inhibitor) 364206*

MMP Inhibitor Set I 444255*

TGFb RI Inhibitor II 616452*

*Available from EMD Chemicals Inc.

For a complete listing of cancer-related products from Millipore, please visit www.millipore.com/cancer.

For a complete listing of cancer-related products from EMD Chemicals, please visit www.emdbiosciences.com.

Use our easy online tool to design your MILLIPLEX map kits. Simply choose the species, panel, and analytes that best fit your needs, and EMD Millipore will build the kit to your specifications. Visit www.millipore.com/kits.

30

Related Research Support ToolsEnsure that your discoveries have the highest impact and biological relevance by starting with quality cell

and tissue samples. EMD Millipore’s sterile filtration tools, cultureware, and automated cell counting

system enable robust, uniform, contamination-free cell and tissue culture.

Sterile FiltrationEliminating contaminants from your cell growth media and additives is absolutely crucial to preserving the

integrity and accuracy of your cell cultures. EMD Millipore’s comprehensive line of sterile filtration tools have

been specifically designed to address these needs and to ensure the reproducibility of your cancer research.

vacuum-Driven Sterile FiltersStericup® filter devices combine a filter unit with a receiver flask and cap for processing and storage.

Description Membrane/Application Pore Size (µm)Funnel Capacity (mL) Receiver Bottle Catalogue No.

Stericup-GP Filter Units Millipore Express PLUS (PES) / fast

filtration of tissue culture media and

buffers

0.22 500 500 mL SCGPU05RE

Cultureware

Description No. of Layers Total Surface Area (cm2) Qty/Pk Catalogue No.

Millicell HY FlaskSTEM CELL TESTED

3 600 16 PFHYS0616

5 1000 8 PFHYS1008

Description System Components Membrane/Pore Size Qty/Pk Catalogue No.

Millicell-96 cell culture insert plates

96-well cell culture plate, single-well feeder tray and lid Isopore (Polycarbonate) 5 PSHT004R5

96-well cell culture plate, 96-well receiver tray and lid Isopore (Polycarbonate) 5 PSHT004S5

Description Qty/Pk Catalogue No.

Millicell EZ SLIDE (4-well glass) 16 PFHYS0616

8 PFHYS1008

Millicell EZ SLIDE (8-well glass) 16 PEZGS0816

96 PEZGS0896

Millicell EZ SLIDE Microscope Slide Holder 1 PEZXMSH01

Millicell Membrane-Based Cell CultureFor more relevant cell-based assays, try growing your cells on EMD Millipore’s membrane-based cell culture

products. The optimized membranes result in cells with structure and function that more closely mimic what

occurs in vivo. Obtain high quality results for screening, cell signaling assays, proliferation studies, and more.

Millicell HY (High-Yield) Cell Culture FlasksSimplify your cell culture. Obtain robust cell growth for your next big experiment by using

Millicell HY multilayer flasks to save time, incubator space, reagents, and money.

Millicell EZ SLIDESUse the Millicell EZ SLIDE to culture, fix, stain and view your cells all in one device. There’s no need to tediously move

cover slips from your culture dish to a slide. Leave the wells intact to fix and stain and acquire data simply and quickly

with Millicell EZ SLIDES.

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HALLMArkS oF CAnCEr

Self-sufficiency in proliferation signals

Insensitivity to proliferation inhibiting signals

Evading apoptosis

Limitless replicative potential

Sustained angiogenesis

Metabolic reprogramming

Escape from immune control

Tissue invasion and metastasis

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