ToxicityAntibodies, Kits, Assays and Services

Data SheetProduct Selection Guide

THE EXPERTISE OF UPSTATE®, CHEMICON® & LINCO®

IS NOW A PART OF MILLIPORE

As a tools and services provider, EMD Millipore is committed to the advancement of life science research and therapeutic development. As a partner in your research, we offer innovative products, technical support and in-house services. 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. We are here to advance life science together with you.

Platforms, Technologies and Services

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

Upstate® and Chemicon®, we provide validated products with breadth and depth in major research areas

backed by excellent service and support.

Cell Based Assays and High Content AnalysisEMD 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. We also offer an

array of assays for high content multiparametric 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. FlowCellectTM

reagents and kits 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® Multiplex AssaysMILLIPLEX 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. We now offer two multiplex immunoassay

formats for use with Luminex® xMAP® technology: MILLIPLEX MAP and MILLIPLEX MAg. Our MILLIPLEX MAg

kits offer the same benefits as our MILLIPLEX MAP kits, as well as full-plate washing for higher throughput

and the flexibility to use traditional vacuum methods or convenient magnetic bead washers. The MILLIPLEX

platform enables the simultaneous detection of multiple soluble or intracellular biomarkers. These flexible and

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

dynamic range.

Calbiochem® CompoundsCalbiochem high quality inhibitors, biochemicals, antibodies, proteins and kits have been cited in thousands of

peer-reviewed publications. Biochemical and environmental signals control intracellular processes as well as

interactions between cells, tissues and organs. As a result, 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. In fact, many drug candidates are

enzyme inhibitors. 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.

Services EMD 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.

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The Relevance of Toxicity StudiesToxicity studies are critical to all stages of a fully integrated drug

development program and are used to augment the interpretation of

absorption, distribution, metabolism and excretion (ADME) results. As

toxicity has been found to be the leading cause of drug failure, research

goals are to establish sensitive, rapid methods for determining organ-

specific damage as quickly as possible. From cells to organs and tissues

to animals, researchers are using a broad range of in vitro and in vivo

assays, everything from classic immunochemistry and flow cytometry

to multiplexed assays and high content analysis in order to answer their

biological questions.

Treating cells with cytotoxic substances can result in a variety of cell

fates, including oxidative stress, necrosis, apoptosis or growth arrest.

There is increasing evidence that oxidative stress generates excess

free radicals, which damage biomolecules, leading to specific and diverse

diseases.

Toxicity can affect one or more systems in the body; the major classes of

toxicity are:

• Neurotoxicity: affecting the brain, spinal cord or peripheral nervous

system

• Cardiotoxicity: affecting the heart or vasculature

• Hepatotoxicity: affecting the liver

• Nephrotoxicity: affecting the kidneys

Advances in Toxicity TestingImprovements in in vitro cell culture have given researchers a viable

alternative and/or complement to live animal testing. In vitro assays are

being used earlier in toxicity testing pipelines, often to assess risks or set

up controls. In vitro models can also enable researchers to interpret the

mechanism behind a toxic response sooner than by inspection of a live

animal.

The expansion of small molecule libraries available for research use has

accelerated toxicity testing studies by revealing relationships between

chemical structures and toxic effects. By using inhibitors of specific

pathways, such as apoptosis, hypoxia, cell cycle, or DNA damage signaling,

for example, drug developers can block particular pathways and determine

if toxic response is affected.

This Product Selection Guide contains information on forms of toxicity and

featured assays, kits, inhibitors and services for studying them. Together

with our customers, EMD Millipore is evolving the science of toxicity

testing, providing the products, services and leadership that advance

toxicology research, at every stage of drug discovery. While cost and speed

are critical, biological relevance is just as vital; our customers work with us

to stay on the cutting edge.

Table of Contents

NEUROTOXICITY 4

MILLIPLEX MAP Human Neurodegenerative Disease Panels

Phosphorylated Neurofilament (pNF-H) Sandwich ELISA Kit

Neurite Outgrowth as a Measure of Neurotoxicity

FEATURED PRODUCTS:Calbiochem Cholinesterase and Amyloidogenesis Inhibitors

CARDIOTOXICITY 10

PrecisION® Recombinant Ion Channel Cell Lines

PrecisION Recombinant hERG Potassium Ion Channel Membrane Preparation

MILLIPLEX MAP Rat Cardiovascular Disease (CVD) Panel 1

Cardiac Stem Cell Isolation Kit

FEATURED PRODUCTS:Calbiochem Ionophores and Calcium Channel Modulators

HEPATOTOXICITY 14

Hepatotoxicity Assay, Human HepG2 Cells

MILLIPLEX MAg Human Liver Protein Magnetic Bead Panel

Anti-Cytochrome P450 CYP450 1A2

NEPHROTOXICITY 17

MILLIPLEX MAg Human Kidney Toxicity Panel 4

MILLIPLEX MAP Rat Kidney Toxicity Panel 1

Anti-Lipocalin-2 (LCN2)

CELL HEALTH 21

Flow Cytometry Kits and Instrumentation

ToxReporter™ Cell Lines and ELISAs

FEATURED PRODUCTS:Calbiochem Lipopolysaccharides and DNA and RNA Polymerase Inhibitors

OXIDATIVE STRESS 29

Manganese Superoxide Dismutase (MnSOD) and Histone H2A.X Phosphorylation Assay

Assays for the Detection of Oxidized Proteins

Nitrotyrosine ELISA kit

FEATURED PRODUCTS:Calbiochem Nitric Oxide Synthase, Arginase and Glutathione S-Transferase (GST) Inhibitors

TOXICITY SERVICES 34

Lead Discovery Services

Bulk and Custom Services

BioPharma Services

Introduction

4

NeurotoxicityDue to the sensitive homeostatic metabolism of neural cells, they are extraordinarily susceptible to toxicity. Accordingly, drug candidates for many neurological and systemic diseases need to be screened for toxicity early in the discovery process. Traditional in vitro neurotoxicity assays have limitations, including a lack of neuronal-specific markers, restriction to single endpoint readouts, sensitivity to only late-stage lethality and poor amenability to scale-up. Neuronal outgrowth and morphology analysis has further been hampered by lack of platforms for neurite isolation in vitro as well as limited ability to assess tissue-wide damage. These challenges impact drug development, where knowing as much as possible about compounds in advance is critical for avoiding unexpected, adverse effects during clinical trials. EMD Millipore’s products and services exploit multiplex detection of neural biomarkers and isolation of neural architecture for the purpose of analyzing neural development, function, dysfunction and toxicity.

MILLIPLEX map Human Neurodegenerative Disease Panels(Catalogue Nos. HNDG1-36K, HNGD2-36K, HNDG3-36K)Neurodegeneration is caused not only by targeted diseases,

but it can also be caused by toxicity, inflammation or

autoimmune disorders. Explore all the possibilities with

our neurodegenerative disease panels. We are the first to

provide multiplex kits for the study of neuroscience. The

Luminex xMAP technology-based MILLIPLEX MAP neuroscience

panels help you gain a deeper understanding of the

complexities of the nervous system.

• This kit may be used for the analysis of all or any combination of the analytes in this panel in serum, plasma, CSF (cerebrospinal fluid), and cell/tissue extract or culture samples.

• This is a 3.5 hour assay using 25 µL or less of sample.

• Recommended dilution for serum and plasma is 1:40,000 and recommended dilution for CSF is 1:400.

Concentration (ng/mL)

xxxx

x

xxxI

xI

xI

xIxI

xI xI

I

I

I

I

II

I

0.001 0.01 0.1 1 10 100 1,000 10,000

100,000

10,000

1,000

100

10

MILLIPLEX MAP

Human Neurodegenerative Disease Panel 1Standard Curves

Panel 1

Apolipoprotein A1

Apolipoprotein CIII

Apolipoprotein E

x Prealbumin

xI Complement Factor H

Complement C3

I α2 Macroglobulin

Med

ian

Fluo

resc

ence

Inte

nsit

y (M

FI)

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• This kit may be used for the analysis of all or any combination of the analytes in this panel in serum, plasma, CSF (cerebrospinal fluid), and cell/tissue extract or culture samples.

• This is an overnight assay using 25 µL of sample.

• Recommended dilution for serum and plasma is 1:2,000 and recommended dilution for CSF is 1:20.

Concentration (ng/mL)0.001 0.01 0.1 1 10 100 1,000 10,000

100,000

10,000

1,000

100

10

MILLIPLEX MAP

Human Neurodegenerative Disease Panel 2Standard Curves

Panel 2

CRP

α1-Antitrypsin

PEDF

x SAP

xI MIP-4

Complement C4

Med

ian

Fluo

resc

ence

Inte

nsit

y (M

FI)

xx

x

x

xx

x

xI

xI

xI

xI

xIxI xI

• This kit may be used for the analysis of all or any combination of the analytes in this panel in serum, plasma, CSF (cerebrospinal fluid), and cell/tissue extract or culture samples.

• This is an overnight assay using 25 µL of sample.

• Recommended dilution for serum and plasma is 1:100 and sample is used neat with CSF.

Concentration (ng/mL)0.001 0.01 0.1 1 10 100 1,000 10,000

100,000

10,000

1,000

100

10

MILLIPLEX MAP

Human Neurodegenerative Disease Panel 3Standard Curves

Med

ian

Fluo

resc

ence

Inte

nsit

y (M

FI)

Panel 3

xI

x

x

x

x

x

xxIxI

xI

xI

xIxI

NCAMPDGF-AB/BB

RANTES

PAI-1 (total)

x MPO

xI Cathepsin D

PDGF-AA

sVCAM-1

BDNF

slCAM-1

10080604020

Con Plasma

P value=0.007

AdPlasma

0

C3 10080604020

Con Plasma

P value=0.005

AdPlasma

0

sICAM-1

1500

1000

500

Con CSF

P value=0.038

Ad CSF0

APO A11500

1000

500

Con CSF

P value=0.003

Ad CSF0

Prealbumin252015105

Con CSF

P value=0.021

Ad CSF0

SAP

2000

1500

1000

50

Con

CSF

Ad C

SF

P value=0.006 P value=0.002

0

250200

100150

500

A1AT

Con

Plasm

a

Ad P

lasma

200

150

100

50

Con Plasma

P value=0.028

AdPlasma

0

C4

0.04

0.03

0.02

0.01

Con

CSF

Ad C

SF

P value=0.009 P value=0.018

0.00

15

10

5

0

PDGF-AA

Con

Plasm

a Ad

Three human neurodegenerative disease multiplex kits (HNDG1-36K, HNDG2-36K and HNDG3-36K) were used to measure samples from both normal subjects (Con) and Alzheimer's disease (Ad) patients. Significantly different biomarker levels were detected between these two groups.

Relative Differences Between Normal and Ad Samples: Plasma Relative Differences Between Normaland Ad Samples: CSF and Plasma

Relative Differences Between Normal and Ad Samples: CSF

6

Phosphorylated Neurofilament (pNF-H) Sandwich ELISA Kit (Catalogue No. NS170)A Sensitive Biomarker of Axonal InjuryThe Phosphorylated Neurofilament H ELISA kit can detect

pNF-H in the sera of animals which have had spinal cord and

brain injuries, though no pNF-H can be detected in the sera

of uninjured animals. Levels of pNF-H may peak at more than

250 ng/mL in serum and return to zero in the weeks following

injury. Since pNF-H is only expressed in axons, the detection

of serum pNF-H is a convenient and sensitive biomarker of

the extent of axonal injury. The assay works on samples from

all mammalian species tested to date, including samples from

rat, mouse, rabbit, feline, porcine, bovine and human.

ng/m

L pN

F-H

Blood pNF-H in G93A SOD1

Age in Days

0

10

20

30

40

60 70 80 90 100 110 120 130

Measurement of pNF-H using Cat. No. NS170 in transgenic mouse for human copper/zinc superoxide dismutase 1 G93A (SOD1 with incorporated G93A mutation). This mutant SOD1 (found in some familial forms of ALS) causes a disease state in the mouse very similar to human amyotrophic lateral sclerosis (ALS). 0.5 microliters of plasma was used for the assay. There is a weak signal in most mice at 74 days which increases as the disease progresses. The animals do not show obvious ALS symptoms until about 90 days, suggesting that the assay can clearly detect presymptomatic axonal loss.

Neurite Outgrowth as a Measure of NeurotoxicityThe critical process of neurite outgrowth is easily affected by neuronal health and neurotoxicity. The following are three

unique solutions for the measurement of toxin effect on neuronal extension.

Solution 1: Neurotoxicity and Neurite Outgrowth Assay for Quantitative Cell Imaging (QCI) (Catalogue No. HCS220)The Neurite Outgrowth QCI assay is immunofluorescence-

based, and uses a high quality primary antibody that

specifically labels neurites and neuronal cell bodies from a

wide variety of mammalian species, including human, mouse

and rat. The reagents and protocols contained within the kit

provide a complete, fast, efficient solution for quantifying

neurite outgrowth.

Our Neurotoxicity QCI kits offer enhanced sensitivity,

neuronal specificity and the capability to detect multiple

modes of neuronal damage. These assays include assessment

of cell number, neuronal- and glial-specific markers.

Neurotoxic properties of K252a

Rat PC12 cells were treated with 0.4% DMSO (panel A) or 1 μM K252a (panel B) for 96 h during NGF-induced differentiation. Cells were then immunostained and imaged to identify the effects on neurite outgrowth and synaptic vesicle formation. Images show Hoechst nuclear stain (blue), β III-tubulin (green) (from Cat. No. HCS220) and synaptophysin (red) (from Cat. No. HCS226).

B.

A.

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Visualization of Neurite Growth:

N1E-115 cells demonstrate neurite outgrowth with excellent signal-to-noise, using the neurite outgrowth assay plus kit (Cat. No. NS230). Neurites are absent from BSA-coated inserts (top) while laminin-coated inserts clearly show neurite outgrowth via staining with the included 10X Cell Staining Solution (bottom).

Solution 2: Neurite Outgrowth Assay Plus Kit (Catalogue No. NS230)Recent research has focused on studying the causes of

directionality of axon growth. The polarity of axon growth

coincides with gradients of extracellular signals, but it is

not yet clear how extracellular gradients translate into

asymmetric distribution and function of intracellular proteins

driving neurite outgrowth. To understand the biochemical

mechanisms by which axons grow in response to signaling,

one should assay each axon, in isolation from somas and

other neurons.

Timecourse Analysis

0h 1h 6h 24h 48h0

OD

45

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

Laminin BSA

Laminin, but not BSA, supports outgrowth of neurites from N1E-115 cells over time, as determined using the neurite outgrowth assay plus kit (Cat. No. NS230). Neurite extension was analyzed after fixing cells at the time points indicated (from 0 to 48 hours post-seeding onto inserts). The amount of signal detected for the laminin-coated inserts dramatically increased over time, while the BSA control was largely unchanged.

N1E-115 cells clearly demonstrate neurite outgrowth through the AXIS channels (150 μm) using the Milli-Mark™ FluoroPan neuronal marker (MAB2300X) shown in green, versus DAPI (blue).

Solution 3: AXIS™ Axon Isolation Device (Catalogue No. AX15010)The AXIS axon isolation device is a two chamber system,

each composed of two wells and an interconnected channel,

each of which is separated by a set of microgrooves. The

hydrostatic pressure formed by volume differential between

chambers induces fluidic isolation of the solution on the low

volume side of the device. The microfluidic design of the AXIS

device allows for development and maintenance of a fluidic

gradient of chemoattractants, toxins or other molecules of

interest, facilitating controlled exposure and differentiation

of axons.

AXIS microfluidic design:

MICROGROOVES

CH

AM

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Calbiochem Cholinesterase and Amyloidogenesis InhibitorsCholinesterase InhibitorsUnder normal conditions, extensive inhibition of

acetylcholinesterase (AChE) leads to excess synaptic

acetylcholine levels, over-stimulation of cholinergic

receptors, alterations of postsynaptic cell function and

consequent signs of cholinergic toxicity. Cholinesterase

inhibitors thus exhibit both pharmacological and

toxicological mechanisms of action.

A large number of autonomic neurons are cholinergic

in nature. Cholinergic terminals contain a large number

of small acetylcholine (ACh)-containing, membrane-

bound vesicles concentrated near the synaptic end.

Following their release from the pre-synaptic end,

ACh molecules activate cholinoreceptors on the post-

synaptic membrane. AChE is a tetrameric protein that

catalyzes the hydrolysis of acetylcholine. The active

site of AChE includes a serine hydroxyl group that is

rendered more nucleophilic through the proton-acceptor

action of a nearby histidine residue. The serine residue

exerts a nucleophilic attack on the carbonyl carbon

of acetylcholine. AChE inhibitors may act by either

competitively blocking hydrolysis without reacting

with the enzyme, or may acylate the serine hydroxyl

group, forming a carbamyl ester, which is more stable

than acetate and is less likely to abandon the active

site of the enzyme. AChE inhibitors, which increase

the availability of acetylcholine in central synapses,

as well as muscarinic agonists, have become the main

approach to symptomatic treatment of patients with

Alzheimer’s disease (AD). These agents do not reverse

the progression of the disease, but they do contribute to

modest improvements in memory, thinking and reasoning

skills in AD patients.

Amyloidogenesis InhibitorsResearch in the area of trafficking and processing of

amyloid precursor proteins provides additional insights

into amyloid precursor protein biology and neuronal

apoptosis as a consequence of increased amyloid peptide

production.

Aβ (β-amyloid peptide) is a major component of

neuritic plaques and cerebrovascular amyloid deposits in

the brains of patients with Alzheimer’s disease (AD). The

cellular origin of amyloid precursor protein (APP) that

gives rise to Aβ is now well understood. Morphological

evidence suggests that APP-immunoreactive neurites,

often capped by Aβ deposits are a major source of

parenchymal amyloid. However, other cells, including

astroglia, microglia and vascular cells, may contribute to

the formation of Aβ. Because a long-standing hypothesis

posits that Aβ deposits are neurotoxic and are causative

factors in the development and progression of AD,

development and use of inhibitors of Aβ fibrillogenesis

are pivotal to neurotoxicity research.

CH

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S

Muscarinic Effects

CardiovascularVasodilation Reduced Cardiac Rate Reduced Force of Contraction

GastrointestinalIncreased PeristalsisEnhanced Secretary ActivitySphincter Relaxation

Granular SecretionsIncreased Pancreatic SecretionsEnhanced Salivary K and Water SecretionIncreased Lacrimal SecretionsIncreased Adrenal Medullary Secretions

Urinary BladderIncreased Ureteral PeristalsisReduced Bladder Capacity

Nicotinic Effects

CNS EffectsStimulation of CNSExcitation of Respiration

Autonomic GangliaExcitation of Sympathetic andParasympathetic Ganglia

Neuromuscular JunctionsMuscle Contraction

FEATURED PRODUCTS

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Antibodies

AB5864 Anti-Myelin Basic Protein

AB5611 Anti-Neuroketal

MAB5328 Anti-RAGE

AXIS

AX45005 AXIS Axon Isolation Device, 450 μm

AX45010 AXIS Axon Isolation Device, 450 μm

AX50010 AXIS Axon Isolation Device, 500 μm

AX90010 AXIS Axon Isolation Device, 900 μm

Calbiochem Inhibitors

30967 Diisopropylfluorophosphate

345670 Galanthamine, Hydrobromide

385885 (±)-Huperzine A

171581 Aβ40 Fibrillogenesis Inhibitor

171587 Aβ42 Fibrillogenesis Inhibitor II

171588 Aβ42 Fibrillogenesis Inhibitor III

233165 Clioquinol

287840 Diclofenac Sodium

344079 Flurbiprofen

345834 Genistein, Soybean

554325 Resveratrol

ELISAs

EZHS40 High Sensitivity Human Amyloid β40

EZHS42 High Sensitivity Human Amyloid β42

EZHS-SET High Sensitivity Human Amyloid β40 and 42 ELISA Set

EZBRAIN40 Human Amyloid β40 Brain ELISA

EZBRAIN42 Human Amyloid β42 Brain ELISA

EZBRAIN-SET Human Amyloid β40 and 42 Brain ELISA Set

NS400 a-Synuclein ELISA Kit

NS690 Amyloid Precursor Protein (APP) ELISA Available Oct., 2010

NS830 Glial Fibrillary Acidic Protein (GFAP) ELISA Available Oct., 2010

Lysates

CL102 Rat Brain

12-144 Microsomal Preparation, rat brain

Multiplex Kits

HBDP-33K MILLIPLEX MAP Human Brain-Derived Protein Panel

HNP-35K MILLIPLEX MAP Human Neuropeptide Panel

HPT-66K MILLIPLEX MAP Human Pituitary Panel

RPT86K MILLIPLEX MAP Rat Pituitary Panel

RSH69K MILLIPLEX MAP Rat Stress Hormone Panel

HNDG4-36K MILLIPLEX MAP Human Neurodegenerative Panel 4 Available Nov., 2010

HND1MAG-39 MILLIPLEX MAg Human Neurological Disorders Panel 1 Available Nov., 2010

HND2MAG-39K MILLIPLEX MAg Human Neurological Disorders Panel 2 Available Nov., 2010

Quantitative Cell Imaging Kits

HCS221 High Content Analysis Kit for Gliosis

HCS222 High Content Analysis Kit for Co-Culture of Neurons and Astrocytes

HCS226 High Content Analysis Kit for Neurite Outgrowth and Synaptic Activity

Tissue Stains

AG325 Fluoro-Jade® C stain for neural tissue degeneration

AG335 Fluoro-Ruby® stain for acute axonal degeneration

OTHER KEY NEUROTOXICITY PRODUCTS:

Visit our website for all toxicity-related products and services.

10

CardiotoxicityCardiovascular toxicity encompasses cardiotoxicity, or damage to the heart, and vascular disorders, such as atherosclerosis and thrombosis. Cardiotoxicity has received heightened emphasis due to findings that noncardiovascular drugs can carry a risk of rare but life-threatening arrhythmias. This has resulted in relabeling or withdrawal of major drugs, such as terfenadine, astemizole, grepafloxin and cisapride.

The most common arrhythmia caused by multiple drug classes is a ventricular tachyarrhythmia known as torsades de pointes (TdP). Drug-induced TdP can revert spontaneously without serious symptoms, but it can also cause syncope or sudden death. Studies have revealed that congenital mutations in ion channels are common causes of heart arrhythmias, indicating that cardiotoxic compounds are also likely to be ion channel modulators.

Recent advances in ion channel screening, availability of assay kits, optimized cell lines and instrumentation led the ICH to issue the S7B guidance for industry. These guidelines specifically recommend a safety testing strategy that includes electrophysiology screens on cultured cardiac myocytes or cells expressing cloned human cardiac ion channels. These recommendations, along with advances in ion channel technology, have helped to shape the near future of cardiotoxicity research.

PrecisION Recombinant Ion Channel Cell LinesThe fundamental role of ion channels in both normal and

diseased states has made them targets for drug discovery

in a wide variety of therapeutic areas including pain, cardiac

disease, neurological disorders, obesity and diabetes. New

high throughput functional screening technologies have

created the need for high quality ion channel cell lines

for the accurate assessment of compound activity on

therapeutically-relevant ion channel targets. As a result,

EMD Millipore has built a strong portfolio of ion channel cell

lines to meet these emerging needs in the biopharmaceutical

industry.

Our PrecisION ion channel cell lines have been

pharmacologically and functionally validated—using both

conventional and automated electrophysiology—and are

stable for over 25 cell passages.

PrecisION hERG-CHO Recombinant Cell Line and hERG-HEK Recombinant Cell Line (Catalogue Nos. CYL3038 and CYL3039)The FDA, ICH S7B guidelines set out a non-clinical testing

strategy to assess the effects of pharmaceuticals on

ventricular repolarization and proarrhythmic risk. The most

common mechanism for this is inhibition of the delayed

rectifier potassium current (IKr) mediated by the potassium

ion channel (Kv11.1), encoded by the human ether-à-go-go-

related gene (hERG).

Two examples of our PrecisION cell line are highlighted

here. Millipore has recombinantly expressed the hERG

potassium channel (hKv11.1) in both CHO-K1 (Cat. No.

CYL3038) and HEK293 (Cat. No. CYL3039) cell lines using

superior vector technologies to provide good stability,

expression and current.

Visit our website to learn more about the PrecisION Recombinant Ion Channel Cell Lines.

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PrecisION Recombinant hERG Potassium Ion Channel Membrane Preparation (Catalogue No. CYL4039)The human ether-à-go-go-related gene (hERG) encodes

the potassium ion channel responsible for delayed rectifier

potassium current (IKr). There is a demand for high-

throughput binding assays to rapidly and cost-effectively

identify compounds that interact with the hERG channel.

We provide membranes for hERG radioligand binding

assays. These have been validated using several different

radioligands and provide comparable data to that found in

the literature. Radioligand binding assays are a very high

throughput, useful approach for monitoring potential hERG

liability at the earliest phase of drug discovery. Compounds

identified by this approach are candidates for follow-up

electrophysiological screening.

Ion Channel hERG Membrane

-10 -9 -8 -7 -6 -5 -4

[Compound] Log M

[3H

]-A

stem

izol

e bo

und

(cpm

)

900

800

700

600

500

400

300

200

100

0

Astemizole

Dofetilide

Verapamil

WTCisapride

E-4031

Rank ordering small molecule inhibitors for hERG.hERG Membrane Preparation (10 µg/well) was characterized by evaluating the activity for known hERG small molecule inhibitors in a competition binding assay. The membranes were incubated with 3.0 nM [3H]-Astemizole and increasing concentrations of unlabeled compounds to determine sample activity and rank order.

MILLIPLEX map Rat Cardiovascular Disease (CVD) Panel 1 (Catalogue No. RCVD1-89K)The rat is one of the most common models for

cardiovascular diseases in mechanism studies, new drug

screening and preclinical trials. Levels of soluble biomarkers

such as BNP, Troponin T, TIMP-1, VEGF, MPO and vWF are

important indicators for model establishment, drug in vivo

testing and effect evaluation. Troponin T levels in particular

have been implicated in cardiotoxicity.

MILLIPLEX MAP Rat CVD Panel 1Standard Curves

100,000

10,000

1,000

100

10

0 10 100

1,000

10,00

0

100,0

00

Med

ian

Fluo

resc

ence

Inte

nsit

y (M

FI)

Concentration(ng/mL for MPO and vWF, pg/mL for others)

BNP

TNF-αMCP-1

Tnl

IL-6vWF

• This kit may be used for the analysis of all or any combination of the analytes in this panel in serum, plasma, other body fluids, and cell/tissue extract or culture samples.

• Generally, serum or plasma samples from normal subjects should be diluted 1:4 using the assay buffer provided in the kit as the sample diluent.

• This is an overnight assay requiring 25 µL sample volume.

MPO TIMP-1TnT

PAI-1 (total)

VEGF

Troponin T Normal vs. SHR Serum

0

pg/m

L

50

100

150

200

250

300

350

400

Normal SHR

SHR (Spontaneously Hypertensive Rat) and normal rats were tested with this kit for levels of Troponin T. The SHR rats had higher levels of Troponin T in their serum. Normal serum n=16; SHR serum n=7.

12

Discrete cell populations can be isolated from ventricular heart tissue through differential gradient centrifugation. (A) Representative photos depicting heterogeneous cell populations present in the lower phase before centrifugation and a pure CSC population present in the upper phase after centrifugation. (B) Purity of differential gradient isolated CSCs as determined by flow cytometry analysis for the stem cell marker Sca-1.

Before Centrifugation

After Centrifugation

Cardiac Stem Cell Isolation Kit (Catalogue No. SCR061)The isolation and expansion of cardiac stem cells (CSCs)

opens new opportunities in cardiac regenerative medicine.

CSCs have recently been isolated from human and murine

tissues based on cell biomarkers (Sca-1, TERT, c-Kit, side

population), demonstrating the presence of a non-circulating

stem cell niche within the myocardium. These cells appear to

be bi-potent in their capacity to form cardiomyocytes and

vascular endothelial cells. Preliminary engraftment studies

suggest that these cells are ideal candidates for future

research on cardiac regeneration. However, CSCs are rare

and isolation is challenging. To overcome these obstacles,

we developed an easy-to-use cell isolation kit that is capable

of obtaining a high-yield, pure population of CSCs. This

advancement enables purification of significantly greater

numbers of CSCs for cardiac regeneration and cardiotoxicity

studies without the need for time consuming, complex

protocols and expensive cell sorting equipment.

IonophoresIonophores disrupt transmembrane ion concentration

gradients, required for the proper functioning and

survival of microorganisms. In laboratory research,

ionophores are used to increase the permeability of

biological membranes to certain ions, such as rapidly

raising or lowering intracellular Ca2+ concentrations or

affecting the activity of the Na+/K+ pump, in order to

study the resultant physiological responses.

Ionophores are hydrophobic molecules that selectively

bind to a given metal ion and increase its cell permeability.

The inner part of ionophores is made of polar groups

forming a tetra- or octahedral geometry that fits and

encloses a specific ion. Ionophores shield the charge of

the ion to be transported, enabling it to penetrate the

hydrophobic interior of the lipid bilayer. Ionophores may

be channel-forming ionophores or mobile ion carriers.

Several uncoupling agents, such as 2,4-dinitrophenol and

carbonyl cyanide m-chlorophenylhydrazone, may also act

as H+ ionophores. They act as lipid-soluble weak acids

and provide a pathway for the flow of H+ across the inner

mitochondrial membranes.

Calcium Signaling Products, Including IP3, Ryanodine and Calcium Channel ModulatorsToxicology-related pharmacological, electrophysiological

and biochemical investigations include an evaluation

of calcium channel modulation effects on excitation-

contraction and excitation-secretion coupling processes.

The divalent cation calcium (Ca2+) is used by cells as

a second messenger to control many cellular processes

including muscle contraction, secretion, metabolism,

neuronal excitability, cell proliferation and cell death.

The cell has access to two sources of signal Ca2+, entry

from the external medium and release from internal

stores. These Ca2+ ON mechanisms depend upon Ca2+

entry through channels in the plasma membrane or

Ca2+ release through ryanodine receptors (RYRs) or

inositol trisphosphate receptors (InsP3Rs). These Ca2+

ON mechanisms are balanced by Ca2+ pumps which

constitute the OFF mechanisms responsible for removing

the Ca2+ signal. These ON and OFF mechanisms are often

organized to produce brief spikes and waves of calcium.

Cells may avoid the cytotoxic effects of calcium by

employing this oscillatory mode of calcium signaling.

FEATURED PRODUCTS

B.

A.

Calbiochem Ionophores and Calcium Channel Modulators

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Antibodies

AB1549 Anti-Brain Natriuretic Peptide (BNP)

AB5412 Anti-Calcium Channel, Voltage Gated Cardiac a1C

06-382 Anti-Calsequestrin, cardiac

MAB3458 Anti-Myofibroblasts

06-811 Anti-Na+ Channel a, cardiac (III-IV loop)

05-205 Anti-Phospholamban, clone A1

07-052 Anti-phospho-Phospholamban (Ser16)

AB5930 Anti-Potassium Channel ERG1, C-terminus

AB5932 Anti-Potassium Channel KvLQT1, C-terminus

MAB2636 Anti-SERCA2, clone IID8

MAB1693 Anti-Troponin T, clone 2G3

MAB3150 Anti-Cardiac Troponin I, a.a. 41-49, clone 284 (19C7)

MAB3152 Anti-Cardiac Troponin I, a.a. 87-91, clone 8E10

Calbiochem Inhibitors & Modulators

115500 Adenophostin A, Hexasodium Salt

100065 2-APB

286888 BHQ

203675 Bombesin, Free Base

251680 Dantrolene, Sodium Salt

298711 Diethyl Pyrocarbonate

682160 Xestospongin C, Xestospongia sp.

682162 Xestospongin D, Xestospongia sp.

512743 Pasteurella Multocida Toxin, Pasteurella multocida

Calbiochem Ionophore Related Products

100107 A23187, 4 Bromo

100105 A23187, Free Acid, Streptomyces chartreusensis

100106 A23187, Mixed Calcium-Magnesium Salt

205535 CA 1001

368020 Gramicidin A, High Purity, Bacillus brevis

407952 Ionomycin, Calcium Salt, Streptomyces conglobatus

407950 Ionomycin, Free Acid, Streptomyces conglobatus

475897 Monensin Methyl Ester

475895 Monensin, Sodium Salt, High Purity

481990 Nigericin, Sodium Salt, Streptomyces hygroscopicus

475914 Nystatin, Streptomyces noursei

569385 SQI-Pr

676377 Valinomycin, Streptomyces fulvissimus

Lysates

CL104 Rat Heart

CL304-250UG Human Heart

Multiplex Kits

HCVD1-67AK MILLIPLEX MAP Human Cardiovascular Disease (CVD) Panel 1

HCVD2-67BK MILLIPLEX MAP Human Cardiovascular Disease (CVD) Panel 2

MCVD1-77AK MILLIPLEX MAP Mouse Cardiovascular Disease (CVD) Panel 1

MCVD277BK MILLIPLEX MAP Mouse Cardiovascular Disease (CVD) Panel 2

RCVD2-89K MILLIPLEX MAP Rat Cardiovascular Disease (CVD) Panel 2

RCVD3-89K MILLIPLEX MAP Rat Cardiovascular Disease (CVD) Panel 3

Stem Cell Reagents

SCM101 Cardiac Stem Cell Maintenance Medium

SCM102 Cardiomyocyte Differentiation Medium

OTHER KEY CARDIOTOXICITY PRODUCTS:

Visit our website for all toxicity-related products and services.

14

HepatotoxicityDrug-induced hepatotoxicity is a major factor in both the high fail rate of drug development and withdrawal of drugs from the market. Consequently, it is crucial to identify potential hepatotoxins early in the drug development process. Detection of hepatotoxicity using traditional in vitro studies has been unreliable, due to poor assay specificity, insufficient endpoints and an inability to detect early stages of hepatotoxicity. High-content screening has been demonstrated to be an effective tool for determination of drug-induced human hepatotoxicity using the human hepatocellular carcinoma cell line HepG2, a widely used cellular model for in vitro cytotoxicity studies. Data suggest that high content screening for hepatotoxicity using human HepG2 cells can be a more reliable indicator of human hepatotoxicity than animal models. EMD Millipore offers multiple options for hepatoxicity profiling, including quantitative cell imaging kits for high content screening and Luminex multiplexed bead-based assays.

Hepatotoxicity Assay, Human HepG2 Cells (Catalogue No. HCS100)The hepatotoxicity assay kit for human HepG2 cells provides

multiparametric, quantitative cell imaging analysis of

drug-induced human hepatotoxicity. This multiplexed kit is

comprised of high-quality, validated, automation-compatible

detection reagents and validated protocols for profiling

multiple human hepatotoxicity endpoints. It provides a means

to screen compounds for a broad range of potentially toxic

effects early in the drug discovery process, providing better

information to drive drug development.

Four color pseudocolored image sets of control and paclitaxel-treated (24 hr) HepG2 cells, stained using Cat. No. HCS100. Blue - Nuclei; Green - Microtubules; Red - Mitochondria; Magenta - Phospho-Histone H3. (A) Untreated cells show a well-defined microtubule cytoskeleton and normal mitochondria and nuclei. The image also includes one cell undergoing mitosis (metaphase). (B) When treated with paclitaxel, subpopulations of cells exhibit bundled microtubles, fragmented nuclei and elevated phospho-histone H3 levels.

B.A.

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MILLIPLEX mag Human Liver Protein Magnetic Bead Panel (Catalogue No. HLPPMAG-57K)Liver-secreted proteins play important roles in metabolic

regulation. For example, liver-secreted proteins have been

shown to regulate circulating lipoprotein levels, energy

expenditure, glucose metabolism and fatty acid uptake. In

addition, some liver-secreted proteins may also serve as

biomarkers for liver toxicity, diseases and gastric cancer.

Accurate measurement of liver proteins is critical to obtain

understanding of their biological functions.

Normal serum and chronic kidney disease serum samples were tested using Cat. No. HLPPMAG-57K. The serum levels for each biomarker tested were higher in the chronic kidney disease samples than in the normal samples. Normal samples n=6, chronic kidney disease samples n=13.

pg/m

L

0.00

40.00

20.00

80.00

100.00

60.00

120.00

140.00

Normal Disease

ANGPTL4

Normal Disease

pg/m

L

0.00

0.80

0.60

0.40

0.20

1.00

1.20

1.40 HGF

Normal Disease

pg/m

L

0.00

4.00

2.00

6.00

14.00

12.00

10.00

8.00

18.00

16.00

FGF-23

MILLIPLEX MAG Human Liver Protein PanelStandard Curves

1

10

100

1,000

10,000

100,000

0.01 0.1 1 10 100 1,000 10,000

Concentration (ng/mL)

Med

ian

Fluo

resc

ence

Inte

nsit

y (M

FI)

AFP

ANGPTL3

ANGPTL4

ANGPTL6

HGF

FABP1

FGF-19

FGF-21

FGF-23

• This is an overnight or same day assay requiring 12.5 µL human serum, plasma, and culture samples. Note: When assaying ANGPTL6/AGF it is recommended that serum samples be used.

• Sample dilution is required.

16

Anti-Cytochrome P450 CYP450 1A2 (Catalogue No. AB10089)A family of 50 closely related isoforms, cytochrome P450

(CYP450) enzymes metabolize a large number of chemicals,

including drugs. The liver is enriched in CYP450s, which

metabolize toxic and potentially toxic compounds. Because

the CYP450s are a very diverse family, these enzymes are

capable of oxidizing a wide variety of drugs.

The range of CYP450s expressed varies from one

individual to another. The field of pharmacogenomics is

dedicated to understanding the relationship between

individual genetic profiles and responses to drugs, including

responses to toxicity.

Anti-CytochromeP450 CYP450 1A2

Western Blot Analysis:Baculovirus-expressed recombinant rat CYP450 1A2 was resolved by electrophore-sis, transferred to nitrocellulose, and probed with anti-CYP450 1A2 (Cat. No. AB10089, 1:2000 dilution). Proteins were visualized using a donkey anti-rabbit secondary antibody conjugated to HRP and a chemiluminescence detection system.

Arrow indicates CYP450 1A2 (~58 kDa).

OTHER KEY HEPATOTOXICITY PRODUCTS:

Antibodies

AB10300 Anti-CYP2A6

AB9916 Anti-CYP2b10

AB10080 Anti-CYP450, clone 2E1

AB10088 Anti-CYP450 1A1

MAB10111 Anti-CYP450 4F11, clone F21 P6 F5

AB10324 Anti-CYP450 Pan 2C

FCMAB115F Anti-TRA-1-60, clone TRA-1-60 FITC conjugate

MAB4349 Anti-TRA-2-49, Liver/Bone/Kidney Alkaline Phosphatase, clone TRA-2-49/6E

MAB4354 Anti-TRA-2-54, Liver/Bone/Kidney Alkaline Phosphatase, clone TRA-2-54/2J

MAB5324 Anti-Polysialic Acid-NCAM, clone 2-2B

AB10339 Anti-UGT1a1

Blots

TB030 Ready-to-Screen Tissue BLOTS™ Human Liver

Multiplex Kits

HLPP-57K MILLIPLEX MAP Human Liver Protein Panel

Tissue protein extracts and lysates

CL108 Rat Liver

CL208 Mouse Liver

CL308 Human Liver

SCC001 Human Neonatal Liver Cell Suspensions

Visit our website for all toxicity-related products and services.

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NephrotoxicityBecause acute kidney failure has a high mortality rate, kidney toxicity is one of the leading reasons for failure of a drug candidate to advance through the pipeline. Drug-induced damage to kidney cells, or renal toxicity, results from drug excretion. Much research has been devoted to the development of better, more sensitive toxicity tests, because the currently used tests for serum creatinine and blood urea nitrogen (BUN) cannot detect kidney damage until one week after it has already occurred. These existing tests also lack tissue specificity. The Critical Path Initiative issued by the FDA was a call for additional quantitative biomarkers for early detection and tissue localization of kidney toxicity. As more and more biomarkers for kidney function are discovered, EMD Millipore supports toxicity research by making available validated assays for testing these new biomarkers.

MILLIPLEX mag Human Kidney Toxicity Magnetic Bead Panel 4 (Catalogue No. HKTX4MAG-38K)The MILLIPLEX MAg Human Kidney Toxicity Panel 4 is to be

used for the simultaneous quantification of the following

5 human kidney toxicity biomarkers in any combination in

urine: Albumin, β-2-Microglobulin, Clusterin, Cystatin C and

Osteopontin (OPN). Most of these biomarkers are included

in the list from the Critical Path Institute’s Predictive Safety

Testing Consortium (PSTC) and are considered qualified

for particular uses in regulatory decision making for acute

kidney injury (AKI).

100,000

10,000

1,000

100

10

10100

1,00010,000

1,000,000

100,000

10,000,000

Clusterin

Albumin

β2M

Cystatin

OPN (ON)

MILLIPLEX MAG Human Kidney Toxicity Panel 4Standard Curves

Med

ian

Fluo

resc

ence

Inte

nsit

y (M

FI)

Concentration (pg/mL)

• This kit may be used for the analysis of all or any combination of the analytes in these panels in urine.

• Overnight assay.• A 25 µL sample volume of a 1:50 dilution is required.

18

Using Cat. No. HKTX4MAG-38K, researchers tested urine samples from 4 different types of individuals: healthy and untreated

patients, patients being treated with cisplatin, patients with chronic kidney infection, and patients with acute renal failure. The

results are shown below.

MILLIPLEX MAG Human Kidney Toxicity Panel 4 in Urine Samples

0

µg/m

L

20

40

60

80

100

120

140

Albumin0

µg/m

L

200

4000

6000

8000

10000

12000

14000

*

*

Clusterin0

µg/m

L

200

400

600

800

1000

1200

1400

OPN0

µg/m

L

1000

2000

3000

4000

5000

Cystatin C0

µg/m

L

500

1000

1500

2000

2500

3000

B2M

Normal On Cisplatin Chronic Kidney Infection Acute Renal Failure

This assay can detect subtle to significant differences in the biomarker levels between normal humans and patients with compromised kidney function. Healthy/untreated: n=14; patients on Cisplatin: n=7; patients with chronic kidney infection: n=5; patients with acute renal failure n=14.

(Catalogue No. RKTX1-37K)The rat is commonly used as a model in kidney disease

and nephrotoxicity studies. The MILLIPLEX MAP Rat Kidney

Toxicity Panel 1 was developed to aid scientists in the

discovery phase of research. This assay was developed for

the simultaneous measurement of 3 critical kidney toxicity

biomarkers in urine: KIM-1, Clusterin and Osteopontin (OPN).

Two of these three biomarkers are included on the list

from the PSTC and are considered to be qualified for use in

decision making for AKI.

Three groups of normal male rats (Wistar, age 7-10

weeks, n=8) were injected with vehicle, gentamicin (100 mg/

kg), or N-phenylanthranilic acid (NPA, 500 mg/kg) for three

days. The neat rat urine samples were collected at days 1

and 4 after treatment and measured with MILLIPLEX MAP Rat

Kidney Toxicity Panel 1 for Clusterin, KIM-1 and Osteopontin

(see graph next page).

100,000

10,000

1,000

100

10

1

10100

1,00010,000

100,000

1,000,000

Clusterin (2 hr)

KIM-1 (2 hr)

OPN (2 hr)

Clusterin (ON)

KIM-1 (ON)

OPN (ON)

MILLIPLEX MAP Human Kidney Toxicity Panel 1Standard Curves

Med

ian

Fluo

resc

ence

Inte

nsit

y (M

FI)

Concentration (pg/mL)

• This kit may be used for the analysis of all or any combination of the analytes in these panels in urine.

• Overnight or 1-day assay.• A 25 µL neat urine sample volume required.

MILLIPLEX map Rat Kidney Toxicity Panel 1

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In another experiment, the same kit was used to determine sex-dependent and strain-dependent variations in basal levels of

Clusterin, KIM-1, and Osteopontin. Ten males and ten females of two different strains were tested using the MILLIPLEX MAP Rat

Kidney Toxicity Panel 1.

0

ng/m

L

5

10

15

20

25

30

35

40

CLUSTERIN0

pg/m

L

20

40

60

80

100

120

140

160

OSTEOPONTIN0

pg/m

L

200

400

600

800

1,000

1,200

1,400

KIM 1

Data show that basal kidney biomarker levels vary with respect to both rat sex and strain, indicating the importance of using model animals of the same sex and strain in preclinical toxicity studies.

Sprague Dawley Males Sprague Dawley Females Wistar Males Wistar

Data show subtle to significant concentration differences for each analyte across the treatment groups, as well as observed differences between analyte concentrations at day 1 and day 4 post dosing. Traditional biomarkers (total protein and BUN) were also determined as controls (data not shown).

0

pg/m

L

5,000

10,000

15,000

20,000

Vehicle Gentimicin NPA

CLUSTERIN

pg/m

L

0

200

100

300

500

400

600

Vehicle Gentimicin NPA

OSTEOPONTIN

pg/m

L

0

200

150

100

50

250

350

300

400

Vehicle Gentimicin NPA

KIM-1

Day 1 Day 4

20

Anti-Lipocalin-2 (LCN2)(Catalogue No. AB2267)LCN2 (also known as NGAL, or neutrophil gelatinase-

associated lipocalin) is a small protein expressed in

neutrophils and various epithelial tissues, including the

renal proximal tubules. There has been great interest in

this protein as a possible marker for the onset of acute

kidney injury (AKI), especially following cardiac surgery,

the progression of chronic kidney disease (CKD) and the

survival chances of patients with chronic heart failure (CHF).

EMD Millipore’s anti-LCN2 antibody has been validated for

immunohistochemistry or Western blotting.

Antibodies

05-354 Anti-Clusterin a chain (human), clone 41D

05-355 Anti-Clusterin β chain (rat)

05-356 Anti-Clusterin a chain (rat), clone 7A8-E4

CBL209F Anti-β-2 Microglobulin, clone GJ14, FITC conjugated

CBL306 Anti-β-2 Microglobulin, clone C21

CBL307 Anti-β-2 Microglobulin, clone B2

CBL307-K Anti-β-2 Microglobulin, Clone B2 (bulk size)

AB10910 Anti-Osteopontin (human, mouse)

AB1870 Anti-Osteopontin (human, mouse, rat)

MAB3055 Anti-Osteopontin, recombinant protein only, clone 4AA

MAB10212 Anti-Renin, clone F32 VIII C4

ELISAs

EZHRBP4-18K Human RBP4 ELISA

EZMAGP-23K Mouse AGP ELISA

Lysates

12-146 Microsomal Preparation, rat kidney

CL106 Rat Kidney

CL206 Mouse Kidney

Multiplex Kits

HKTX1-38K MILLIPLEX MAP Human Kidney Toxicity Panel 1

HKTX1MAG-38K MILLIPLEX MAg Human Kidney Toxicity Magnetic Bead Panel 1

HKTX2-38K MILLIPLEX MAP Human Kidney Toxicity Panel 2

HKTX2MAG-38K MILLIPLEX MAg Human Kidney Toxicity Magnetic Bead Panel 2

HKTX3-38K MILLIPLEX MAP Human Kidney Toxicity Panel 3

HKTX3MAG-38K MILLIPLEX MAg Human Kidney Toxicity Magnetic Bead Panel 3

HKTX4-38K MILLIPLEX MAP Human Kidney Toxicity Panel 4

RKTX2-37K MILLIPLEX MAP Rat Kidney Toxicity Panel 2

OTHER KEY NEPHROTOXICITY PRODUCTS:

Visit our website for all toxicity-related products and services.

Immunohistochemistry Analysis: Representative lot data. Paraffin-embedded human spleen tissue was prepared using heat-induced epitope retrieval in citrate buffer, pH 6.0. Immunostaining was performed using a 1:200 dilution of Anti- LCN2 (Cat. No. AB2267). Reactivity was detected using the IHC Select® Detection Kit (Cat. No. DAB050).

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Cell HealthAdverse effects to cell health can be early hallmarks of toxicity. For a complete picture of toxicity in any of the systems described above (neurotoxicity, cardiotoxicity, hepatotoxicity and nephrotoxicity), analyzing cell health parameters can provide valuable information about the molecular mechanisms of toxicity involved, as well as the downstream consequences of toxicity in the intact organism, very early in the drug development process. Parameters of cell health include:

• Mitochondrial membrane potential

• Early apoptosis

• Cell size

• DNA damage

• DNA integrity

• Cell cycle arrest

EMD Millipore provides instruments, assays and cell lines to monitor these parameters and reduce drug failure in costly preclinical or clinical studies.

Flow Cytometry Kits and InstrumentationThe guava flow cytometers use patented microcapillary

flow cell technology to deliver complex cell analysis right

on the benchtop. These instruments use smaller samples,

generate less waste, and are easier to use and maintain

than traditional flow cytometers, all while providing superior

analytical power in the most compact format available.

With the capacity to simultaneously measure multiple

parameters on hundreds of individual cells per second, flow

cytometry offers greater speed, precision and detail than

most other methods for cell health analysis.

FlowCellect kits are our proprietary multiparameter flow

cytometry kits for the analysis of cellular events and/or cell

phenotypes. Each kit has unique combinations of directly

conjugated antibodies and/or fluorescent dyes and protein

reporters to monitor changes in protein expression and

posttranslational modification.

Guava ViaCount® Reagent(Catalogue No. 4000-0040)The ViaCount assay provides rapid and reliable

determinations of viability and total cell count on all guava

systems. Precise, accurate assessments can be made with

a wide variety of cell lines, even those with unusual culture

conditions or a tendency to aggregate. A simple no-wash,

mix-and-read procedure, the ViaCount assay accurately

determines absolute total cell counts, viability assessments,

and apoptotic percentages with as little as 20 µL of sample.

Guava ViaCount uses two DNA binding dyes to identify viable, dead and apoptotic cells.

Debris Viable Cells Apoptotic Dead Cells

Forward Low High High HighScatter

Nuclear Neg High High HighStain

Viability Neg Neg Med HighStain

22

Guava Cell Toxicity Assay(Catalogue No. 4500-0230)Small molecules and other therapeutics often cause toxicity

by stimulating the body’s immune system. This toxicity

can be mediated by T cells, natural killer (NK) cells or other

immune cells. Immune cell-mediated cytotoxicity has never

been easier to assess than with the guava cell toxicity assay.

The assay uses a well-characterized cell tracking dye

that is optimized for use on all guava systems. The dye

diffuses freely into cells and is retained within the cell

without affecting cellular function. Because the assay is

both sensitive and reproducible, assay development time is

reduced. The guava cell toxicity assay provides all relevant

statistics, including percentage of target cells killed, effector

and target cell percentages, and whether the cells are alive

or dead.

Target cell is labeled with

cell tracking dye

Target cell is killed by

effector cell

Membrane isdamaged

Dead target cell

Add dead cell dye: 7-AAD

Effector cell

How the Guava Cell Toxicity Assay Works

Target cells are marked with the cell tracking dye and are easily differentiated from effector cells. The 7-AAD dye delineates those target cells that are killed, all within minutes of dead cell dye addition.

(A) Data show labeled K562 target cells after a 4 hour incubation with effector NK cells. (B) The Analysis Results table displays data in an easy to read format and includes % of Target Cells Killed and effector vs. target cell percentages.

A.

B.

Guava Cell Toxicity Assay

Dead Effector cells

CFSE

7-A

AD

Dead Target cells

Live Effector cells

Live Targetcells

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FlowCellect Bivariate Cell Cycle Kit G2/M Analysis (Catalogue No. FCCH025103)Cell cycle phase distributions can be used to assess

cell health and proliferation and studying the potential

mechanism of antineoplastic agents.

Use the FlowCellect bivariate cell cycle analysis kit to

investigate the G2/M phase transition with high accuracy

and confidence. The phosphorylation of histone H3 at

Ser10 correlates with the G2 to M phase transition and is

a prerequisite for chromatin condensation at mitosis. At

the end of mitosis, histone H3 is rapidly dephosphorylated

and remains unphosphorylated throughout the remainder

of interphase. Therefore, phospho-histone H3 (Ser10) is a

reliable, specific marker of M-phase cells.

Cell Cycle Phases:G1 = 57%S = 19%G2 = 15%M = 3%

Discrimination between G2 and M phase cells by measuring the phosphorylation of Histone H3 on Ser10.Histone H3 is constitutively phosphorylated at Ser10 during metaphase.

FlowCellect Bivariate Cell Cycle Kit for DNA Replication Analysis (Catalogue No. FCCH025102)Investigate DNA replication in the S phase with high accuracy

and confidence. The kit includes a directly conjugated Anti-

BrdU Alexa Fluor® 488 antibody plus a DNA dye (propidium

iodide). BrdU incorporation is a widely accepted method

of measuring DNA replication and kinetics of cell cycle

progression. The percentage of BrdU labeled cells is a reliable

estimate of the S phase compartment, and labeled cells can

then be followed through the cell cycle.

G=24% (-BrdU, 1X DNA content)

S=72%(↑BrdU, 1-2X DNA content)

G2/M=4% (-Brdu, 2X DNA content)

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.

24

FlowCellect MitoDamage Kit (Catalogue No. FCCH100106)The FlowCellect MitoDamage Kit includes MitoSense

Red, a fluorescent cationic dye that accumulates in the

mitochondria, and is responsive to mitochondrial potential

changes. It also includes Annexin V conjugated to a green

sensitive dye, CF488A, which binds to phosphatidylserine (PS)

on the surface of apoptotic cells. Additionally, it includes the

cell impermeant DNA intercalator 7-Aminoactinomycin-D (7-

AAD), a dead cell dye. The simultaneous use of the reagents

allows researchers to obtain information on early, mid and

late apoptosis in one simple assay.

Dot plots depicting Jurkat cells stained using the MitoDamage kit.Jurkat cells uninduced (left column), induced to apoptosis with 2 μM staurosporine (center column) or with 50 μM CCCP (right column), then stained using the MitoDamage kit. Plots show the percentage of positive cells for: 1st row: Apoptosis (Annexin V binding) and mitochondrial membrane potential change 2nd row: Cell death and mitochondrial membrane potential change3rd row: Apoptosis and cell death Data reports that 2 μM staurosporine induces apoptosis in Jurkat cells, and that 50 μM CCCP depolarizes the mitochondrial membrane, but neither condition is sufficient for cell membrane permeabilization and death.

Mit

oSen

se R

ed

Annexin V, CF488A

Mit

oSen

se R

ed7

-AA

D

Annexin V, CF488A

Red

2 F

luor

esec

ence

(RD

2-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

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)

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)

Green Fluorescence (GRN-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)

Green Fluorescence (GRN-HLog)

0.10%

93.9% 4.8%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)

0.26%

98.4% 1.3%100 101 102 103 104

100

101

102

103

104

0.02%

Red

2 F

luor

esec

ence

(RD

2-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

(RD

2-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%

7-AAD

100 101 102 103 104

101

102

103

104

Depolarized Cells

Dead Cells

Live Cells

58.1% 0.08%

41.0% 0.8%

Red

2 F

luor

esce

nce

(RD

2-H

Log)

Red Fluoresecence (RED-HLog)

100

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An innovative platform to measure toxicity levels in a variety of pathways using a single ELISAToxReporter cell lines allow the detection of early biomarkers

of toxicity and cellular stress. Examples of stressful

stimuli include compounds in drug discovery and organic

or inorganic molecules potentially requiring environmental

monitoring. ToxReporter cell lines and ELISAs can be used to

detect multiple cellular responses to toxic stimuli that often

lead to compound failure during drug development. These

responses include:

• Cellular stress

• Oxidative stress

• DNA damage

• Immunosuppression

• Heavy metal toxicity

ToxReporter consists of 10 cell lines containing unique

reporter gene constructs. Each contains toxicological

pathway specific response elements downstream of a

common, stable, secreted reporter biomarker. This reporter

molecule is easily detected in blood, urine and tissue culture

medium using the ELISA, making dose response or time

course experiments straightforward and robust.

Using our 10 cell lines and toxicity ELISA, it is now

possible to identify a potential toxicity issue early in the drug

development process and then include that assay in on-going

SAR studies. This will result in a better understanding of

both the structural features that drive toxicity pathways and

the features driving potency and selectivity.

ToxReporter Cell LinesSpecific Response

ElementCatalogue Number,

Cell LineCatalogue Number,

Starter Pack*Potential Toxicity

Mechanism Comment

p21 ECL-001 ECLSP-001 DNA damage p21 is associated with DNA damage.

AP-1 ECL-002 ECLSP-002 Oxidative stress, DNA damage and Apoptosis

Activator Protein-1 is a redox sensitive transcrip-tion factor associated with oxidative stress, DNA damage and apoptosis. Effect is down regulated by activated GR (Glucocorticoid receptor).

GRE ECL-003 ECLSP-003 Immunosuppres-sant

Glucocorticoid Response Element regulates im-munosuppressive and anti-inflammatory activities in multiple physiological systems.

HSP70 ECL-004 ECLSP-004 Cellular stress, Heavy metal toxicity

Heat Shock Protein 70 is a chaperone protein associated with heat shock, metal toxicity and cellular stress responses.

NFKB ECL-005 ECLSP-005 Oxidative stress, DNA damage

A redox sensitive transcription factor associated with oxidative stress, DNA damage and apoptosis. Effect is down regulated by activated GR (Gluco-corticoid receptor).

HRE ECL-006 ECLSP-006 Mitochondrial DNA damage

Hypoxia Response Element associated with hypox-ic stress, DNA damage, especially mitochondrial.

XRE ECL-007 ECLSP-007 Induction of CYP450s

Xenobiotic Response Element is associated with the AR receptor (Aryl hydrocarbon Receptor) which induces the expression of cytochrome P450s (1A1).

ARE ECL-008 ECLSP-008 Oxidative stress Anti-Oxidant Response Element leads to the expression of a number of phase II detoxification genes in response to electrophilic compounds or oxidants.

Hmox1 ECL-009 ECLSP-009 Oxidative stress Heme oxygenase 1 is associated with oxidative stress response.

p53 ECL-010 ECLSP-010 DNA damage p53 Response Element is associated with DNA damage.

*Starter Packs include the Stimulant, Selection Agent and Assay Media SupplementNote: All kits require the ToxReporter ELISA Kit (Catalogue No. ETR-201K).

ToxReporter™ Cell Lines and ELISAs

26

The Science of ToxReporter Virtually all toxic responses are preceded by the transcriptional activation of stress response pathways with many therapeutic responses involving downstream transcriptional events. Consequently, the promoters of genes activated in this manner have the potential to be used as early biomarkers of toxicity response.

Induction ofToxic Response

Pathway

1 - 10 µL Cultured Media

CELL

PotentialToxin

TranscriptionFactor

ResponsivePromoter Secreted

ToxReporter

ToxRToxReporter

ELISA

ToxReporter AP-1 ToxReporter Cell Line(Catalogue No. ECL-002)AP-1 is a redox-state-sensitive transcription factor

associated with oxidative stress, DNA damage and apoptosis.

In this cell line, a series of AP-1-sensitive promoters are

used to drive the expression of the ToxReporter reporter

molecule. This assay format allows for:

• Time course measurements from a single sample.

• Quantitative detection of early signs of oxidative stress,

DNA damage and apoptosis.

Dose Response Curves. ToxReporter AP-1 cells were cultured on a 96-well microtiter plate as quadruplicate samples for 2 days. Cells were stimulated with the appropriate dilutions of TPA for 24 hours. ToxReporter cell levels were assayed using our ToxReporter ELISA kit. For EC50 calculations, non-linear best-fit dose response was used (GraphPad PRISM™ 5.0 software).

0

-14 -12 -10 -8 -6

Tox

Rep

orte

r (p

g/m

L)

Log [TPA] M

600

400

200

AP-1 Dose ResponseEC50 for TPA = 1.5 nM

Tox

Rep

orte

r (p

g/m

L)

Log [TPA] M

-12 -10-11 -9 -8 -6-7

800

600

200

400

0

AP-1 Time Course

*

8 hours 16 hours 24 hours

Time Course Curves for Dose Response. ToxReporter AP-1 cells were cultured on a 96-well microtiter plate in quadruplicate for 2 days, then treated with appropriate dilutions of stimulant for 8, 16 and 24 hours. At each time point, 5 µL of media from each well was removed and frozen at -20 °C. ToxReporter AP-1 cell levels were assayed when samples from all time points were collected. The asterisk represents the optimal time and stimulant concentration.

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FEATURED PRODUCTS

LipopolysaccharidesLipopolysaccharides (LPS) act as the prototypical

endotoxin because they bind the CD14/TLR4/MD2

receptor complex, which promotes the secretion of pro-

inflammatory cytokines in many cell types, but especially

in macrophages. In immunology, the term “LPS challenge”

refers to the process of exposing a subject to an LPS that

may act as a toxin.

Polysaccharides derived from strains of E. coli or

Salmonella stimulate the activity of inducible nitric oxide

synthase, and induce apoptosis in mouse thymus and

swine lymphocytes.

DNA and RNA Polymerase InhibitorsDNA and RNA polymerases ultimately direct the synthesis

of proteins that carry out most biological functions and

are key structural components of cells, with alteration of

their activity resulting in toxicological effects.

Inhibitors of DNA and RNA polymerases are invaluable

tools in both clinical and research settings. The use of

DNA and RNA polymerase inhibitors aids in delineating the

mechanistic aspects of transcription and DNA replication,

in defining structure-function relationships, and in protein

turnover studies. As DNA and RNA polymerases are

among the most attractive drug targets, the knowledge

about these inhibitors, their structures, and their modes

of action provides the basis for design of new drugs/

antibiotics that will be effective against new pathogens

and antibiotic-resistant mutants of known pathogens.

Calbiochem Lipopolysaccharides and DNA and RNA Polymerase Inhibitors

28

Antibodies

MAB4122 Anti-MDR related Protein, clone MRPm6

MAB4140 Anti-MDR3, clone P3 II-26

MAB4163 Anti-MDR1, clone MC57

MAB448 Anti-MDR1, clone 3C3.2

CT01 MTT Cell Growth Assay Kit

CT02 MTT Cell Growth Assay Kit

2210 Cell Proliferation Assay Kit, WST dye; ELISA based

Calbiochem Inhibitors

129935 Actinomycin D, 7-Amino

178273 Aphidicolin

385883 HSV Replication Inhibitor, BP5

491207 Novobiocin, Sodium Salt

557403 RNA Polymerase III Inhibitor

Calbiochem Lipopolysaccharides

437620 Lipopolysaccharide, E. coli J5

437627 Lipopolysaccharide, E. coli O111: B4

437625 Lipopolysaccharide, E. coli O55: B5

437629 Lipopolysaccharide, Salmonella minnesota Re 595

437650 Lipopolysaccharide, Salmonella typhimurium

437628 Lipopolysaccharide, Ultra Pure, Salmonella minnesota R595 (Re)

FlowCellect Kits

FCCH025143 FlowCellect Cell Cycle CheckPoint ATM DNA Damage Kit

FCCH025142 FlowCellect Cell Cycle CheckPoint H2A.X DNA Damage Kit

FCCH100105 FlowCellect MitoPotential Red Kit

FCCH100107 FlowCellect MitoLive Kit

FCCH100109 FlowCellect MitoStress Kit

FCCH100110 FlowCellect Cytochrome C Kit

FCCH025111 FlowCellect Oxidative Stress Characterization Kit

Guava ViaCount Related Products

4500-0110 Guava ViaCount Flex Reagent Kit For Challenging Samples

4700-0050 Guava ViaCount Cell Dispersant Reagent Kit

MAPmatesTM

46-665 Total HIF1a MAPmate

46-607 Phospho HSP27 (Ser78) MAPmate

46-608 Total HSP27 MAPmate

46-613 Phospho JNK/SAPK1 (Thr183/Tyr185) MAPmate

46-618 Total JNK/SAPK1 MAPmate

46-610 Phospho p38/SAPK (Thr180/Tyr182) MAPmate

46-612 Total p38/SAPK MAPmate

Quantitative Cell Imaging Kits

HCS210 Cyclin B1 and Ki-67 assay

HCS211 Phospho-Histone H3 (Ser10) and Cyclin B1 Assay

HCS212 BrdU and Phospho-Histone H3 (Ser10) Assay

HCS213 Brdu and Ki-67 Assay

HCS216 Phospho-Histone H3 and a tubulin Assay

HCS223 p53/ DNA Damage Assay

HCS224 H2A.X Phosphorylation/DNA Damage Assay

HCS225 H2A.X Phosphorylation and p53 DNA Damage Assay

HCS231 p38 MAPK Assay Kit

HCS235 p53 and p21 Assay Kit

HCS236 Cytochrome C Assay

HCS237 c-Jun Activation Assay

OTHER KEY CELL HEALTH PRODUCTS:

Visit our website for all toxicity-related products and services.

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Oxidative Stress Oxidative stress is characterized by an excess of free radical groups, which creates a potentially unstable cellular environment linked to tissue damage, accelerated aging and degenerative disease. Oxidative stress can result from many factors, including exposure to alcohol, medications, poor nutrition, trauma, cold, toxins and over-exercise. There is increasing evidence that free radicals damage biomolecules, leading to several specific and diverse diseases, such as atherosclerosis, cerebral and heart ischemia-reperfusion injury, cancer, rheumatoid arthritis, inflammation, diabetes, aging and neurodegenerative conditions such as Alzheimer’s disease. Reactive oxygen species (ROS), including superoxide, hydroxyl radicals, hydrogen peroxide and singlet oxygen, are formed when cells are exposed to oxidizing agents or ionizing radiation as the result of metabolic processes. These ROS can cause damage to the genome, an early step in the development of cancerous conditions.

Monitoring compound-induced oxidative stress is crucial for integrated drug discovery and development programs. EMD Millipore’s validated immunoassays and kits to detect oxidative stress are valuable for early elimination of potentially carcinogenic compounds from the pipeline.

Manganese Superoxide Dismutase (MnSOD) and Histone H2A.X Phosphorylation Assay (Catalogue No. HCS233)Oxidative stress is a common denominator in many diseases

and environmental insults and can lead to severe cellular

damage and cell death. One of the earliest and clearest

cellular responses to oxidative stress is the induction of

antioxidant defenses. The manganese-containing superoxide

dismutase of the mitochondria (MnSOD) plays an essential

role in oxidative stress protection. Numerous studies have

shown that MnSOD can be induced to protect against pro-

oxidant insults resulting from cytokine treatment, ultraviolet

light, irradiation, certain tumors, amyotrophic lateral

sclerosis and ischemia/reperfusion.

Kit: HCS233Blue: Hoechst nuclear stainGreen: Phospho-Histone H2A.X (Ser139)Red: Manganese Superoxide Dismutase (MnSOD)Cells: HeLa cervical adenocarcinoma

30

Assays for the Detection of Oxidized Proteins

A: -H20

2 / -DNPH

B: +H20

2 / -DNPH

C: -H20

2 / +DNPH

D: +H20

2 / +DNPH

A.

C.

B.

D.

OxyICC™ Oxidized ProteinDetection Kit(Catalogue No. S7350)This kit contains the chemical and immunological reagents

necessary to detect carbonyl groups using fluorescent

immunocytochemistry. The test method involves

chemical derivatization of protein carbonyl groups with

2,4-dinitrophenylhydrazine (DNPH). Proteins thus are

covalently coupled to DNP at their carbonyl sites. The

DNP-derivatized proteins are detected using biotinylated

antibodies that are specific to the DNP moiety. Subsequent

incubation with fluorescently-conjugated streptavidin

enables detection using fluorescence microscopy. The signal

intensity reflects the extent of oxidative stress.

Oxidative modification of proteins by oxygen free radicals and other reactive species such as hydroxynonenal occurs in physiologic and pathologic processes. As a consequence of the modification, carbonyl groups are introduced into protein side chains by a site-specific mechanism. The oxidative stress detection kits enable simple and sensitive immunodetection of these carbonyl groups, which are hallmarks of protein oxidation.

+

Oxidative Stress Detection with OxyBlot™,ELISA, IC, IH, & Flow Cytometry

Oxidative Stress (H2O2, NO, Superoxides etc.)

Protein sample from cell lysate, tissue homogenate, biological fluids.

Anti-DNP conjugated to HRPYYY

DNPHO

H2N

NHNO2

NO2

+

DNP-derivatized protein

NH

N

NO2

NO2

Immunodetection via WB, EIA, IC, IH, FC

Y

NHN

NO2

NO2

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OxyIHC™ Oxidative StressDetection Kit(Catalogue No. S7450)The OxyIHC Oxidative Stress Detection Kit contains the

chemical and immunological reagents necessary to detect

protein oxidation in various tissues from a variety of organs

and animal species. Like the OxyICC kit, this method involves

chemical derivatization of protein carbonyl groups with

DNPH. The DNP-derivatized proteins are detected using

biotinylated antibodies that are specific to the DNP moiety.

Subsequent incubation with biotin-conjugated secondary

antibody, streptavidin-conjugated HRP and development

using a 3,3’ diaminobenzidine (DAB) staining allows

immunohistochemical detection of protein oxidation.

The OxyIHC assay (Cat. No. S4750) identified oxidative stress in the cerebellar cortex of the Alzheimer’s disease transgenic mouse model. Following methacarn fixation, the brain tissues were paraffin-embedded and sectioned. They were then deparaffinized and antigen retrieval was performed according to standard laboratory protocol. Panels A and C are sections from wild type while panels B and D are sections from the transgenic mice. Negative control reactions were performed with the Derivatization Control Solution (panel A and B) and showed minimal DAB reactivity with only hematoxylin staining. Staining with DNPH resulted in immunoreactivity (panel C and D). Panel C shows basal levels of staining in wild type brain tissue. Panel D shows that the Alzheimer’s disease transgenic mice are under increased oxidative stress.

A.

C.

B.

D.

Nitrotyrosine ELISA kit (Catalogue No. 17-376)In addition to adding carbonyl groups to proteins, ROS such

as nitric oxide (NO), superoxide (O2 -), peroxynitrite (ONOO-)

and hydroxyl radical (OH-) can cause nitration of tyrosine

residues. The nitrotyrosine assay kit with chemiluminescence

detection is a competitive ELISA for the quantitation of

tyrosine nitration. The kit includes all required reagents,

including white high binding 96-well plates, nitrated

BSA standard, anti-nitrotyrosine antibody, LumiGLO®

chemiluminescent detection substrate, and wash buffers.

The assay has a wide dynamic range and high precision, as

shown in the graph, making it a valuable tool for the study of

oxidative stress.

Arginine NOS

NO Phox

SOD

02

0N00-

02-

H202+02

sGC

GTP

cGMP

Nitrotyrosine/Protein

Citrulline

Nitrotyrosine ELISA

No Co

mpetit

or 0.1 1 10 100 1,000 10,000

Nitrated BSA (µg/mL)

CPS

x 1

06

9.0

8.0

7.0

6.0

5.0

4.0

3.0

2.0

1.0

0

The linear range of the nitrotyrosine ELISA encompasses two orders of magnitude.

32

FEATURED PRODUCTS

Calbiochem Nitric Oxide Synthase, Arginase and Glutathione S-Transferase (GST) InhibitorsNitric Oxide Synthase (NOS) InhibitorsLow levels of nitric oxide (NO) produced by the

endothelial (eNOS) and neuronal (nNOS) enzymes

are crucial for signaling, including vasodilatation,

thermoregulation and neuroprotection. High levels of

NO are produced “on-demand” by the inducible (iNOS)

enzyme, to help kill tumors, bacteria and viruses. Both

underproduction and overproduction of NO have been

linked to various human pathologies.

Nitric oxide (•NO), synthesized from L-arginine by the

action of NOS, is a highly reactive, diffusible and unstable

radical, and plays an important role in the regulation

of a wide range of physiological processes, including

cellular immunity, angiogenesis, neurotransmission and

platelet aggregation. NOS is known to exist in three

isoforms which are involved in various aspects of signal

transduction. NOS inhibitors have gained prominence

in the management of ischemic reperfusion injury,

hypotensive effects of drugs and inflammatory response

to cytokines.

Arginase InhibitorsArginase is crucial in the modulation of NO production

under inflammatory conditions (NO synthesis by NOS2),

but it might also play an important role in constitutive

synthesis of NO.

Arginase, existing in two isoforms, plays a significant

role in the regulation of nitric oxide (•NO) synthesis. Due

to the reciprocal regulation between arginase and nitric

oxide synthase, arginase inhibitors are considered to

have therapeutic potential in treating NO-dependent

smooth muscle disorders, such as erectile dysfunctions

and polyamine-induced bronchial constriction.

Lipid Peroxidation

Membrane Damage

NP-SH Oxidation

DNA Breaks

NOSL-Arg

L-Cit

cGMP

GTP

cGMP-GatedIon Channel

cGMP-PDE

PKGActivation

EC Proliferation/Migration

Anglogenesis

Vasodilation Blood Flow

GCFe2+

O½

Base Damage

Apoptosis

Cytostasis

TumorRegression

TumorProgression

0N00-

NO

Glutathione S-Transferase (GST) InhibitorsGlutathione S-transferases (GSTs) are cytosolic enzymes

that catalyze the conjugation of glutathione with a

variety of exogenous and endogenous electrophiles,

and are well characterized members of the general

xenobiotic detoxification system within cells. Inhibitors

of GST are used as both pharmacological tools as well as

potential therapeutics.

GSTs constitute a family of phase II detoxification

isozymes that catalyze the conjugation of glutathione

with a number of hydrophobic compounds. They provide

protection to mammalian cells against the toxic and

neoplastic effects of electrophilic metabolites of

carcinogens and reactive oxygen species. GST status

may be a useful prognostic factor to determine the

clinical outcome of chemotherapy.

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Antibodies

AB1284 Anti-Heme Oxygenase1

AB5480 Anti-SOD1

AB5830 Anti-8-Hydroxydeoxyguanosine

AB9328 Anti-Thioredoxin 1

MAB3560 Anti-8-Oxoguanine

MAB5382 Anti-HIF-1 a

Calbiochem Inhibitors & Modulators

56766 Spermidine, Trihydrochloride

288500 DL-a-Difluoromethylornithine, Hydrochloride

300260 Diphenyleneiodonium Chloride

311204 NG,NG’-Dimethyl-L-arginine, Dihydrochloride

466220 S-Methylisothiourea, Sulfate

483120 NG-Nitro-L-arginine

483125 NG-Nitro-L-arginine Methyl Ester, Hydrochloride

490075 Nitric Oxide Synthase Inhibitor Set

567300 SKF-525A, Hydrochloride

691550 Zinc (II) Protoporphyrin IX

100050 1400W

154500 Aminoguanidine, Hemisulfate

197900 BEC, Hydrochloride

205546 Caffeic Acid

265005 Dexamethasone

311203 NG,NG-Dimethyl-L-arginine, Dihydrochloride

341180 2-Ethyl-2-thiopseudourea, Hydrobromide

400600 L-N5-(1-Iminoethyl)ornithine, Dihydrochloride

444600 MEG, Hydrochloride

472804 S-Methyl-L-thiocitrulline, Dihydrochloride

475886 NG-Monomethyl-L-arginine, Monoacetate Salt

482100 L-NIL, Dihydrochloride

483400 7-Nitroindazole

490070 Nitric Oxide Synthase, Neuronal Inhibitor I

548000 1-Pyrrolidinecarbodithioic Acid, Ammonium Salt

589411 L-Thiocitrulline, Dihydrochloride

ELISA & Western Blot Kits

S7150 OxyBlot Protein Oxidation Detection Kit

S7250 OxyELISATM Oxidized Protein Quantitation Kit

Quantitative Cell Imaging

HCS232 Manganese Superoxide Dismutase (MnSOD) Assay

HCS234 p21 Detection Assay for High Content Screening

OTHER KEY OXIDATIVE STRESS PRODUCTS:

Visit our website for all toxicity-related products and services.

34

Toxicity ServicesProviding critical early assessment across a range of targets, EMD Millipore’s safety and toxicity testing services are more predictive and biologically relevant, empowering you to make better decisions.

With ever-increasing costs of internal drug discovery and development efforts, you can benefit by counting on us to deliver the data you need at any phase in toxicity testing, from in vitro assays all the way through clinical studies.

Use our screening services to assess potential off-target liabilities across a range of target classes. When you know everything you can about your drug candidates—including potential off-target liabilities—you’ll avoid costly errors and develop effective compounds faster.

Safety & Liability Screening PanelDrug Discovery Safety & Liability Screening Panels address

a critical need for early liability screening by providing

industry-leading, functional profiling against 125 GPCR,

kinase, ion channel and phosphatase targets. These targets

were specifically chosen for their relevance to critical

diseases and key pathways, including:

o Neurotoxicity

o Cardiac function

o Immunoprotection

o Diabetes

o Inflammation

o Gastrointestinal liability

Ion

Cha

nnel

% Inhibition

0 20 40 60 80 100

hKir2.1

hKv4.3/hkChIP1

hCav1.2

hCav1.2

hKCNQ1/hminK

hHCN4

hKv1.5

hERG

BIRB0796 Inhibition of Cardiac Ion ChannelsBIRB0796 was developed as a kinase inhibitor to treat chronic inflammation. Its development was halted during clinical trials due to reported hepatotoxicity. To investigate other potential off-target effects, this compound was assayed at 10 μM against cardiac ion channels included in the Safety & Liability Screening Panel. Results indicate significant inhibition of hERG and hKv1.5 currents, indicating potential cardiotoxicity.

5 0

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CardiacProfiler™ ServiceCardiacProfiler is a comprehensive cardiac safety panel that

includes each of the key cardiac channels, providing a robust,

cost-effective alternative to complement more complex

and low throughput assay formats. Our CardiacProfiler

service is fully flexible, enabling you to submit any number of

compounds against any of the CardiacProfiler ion channels.

With our CardiacProfiler service, you can uncover

potential cardiac liability of lead series earlier in the drug

discovery process with high quality functional data within a

1-4 week turnaround time.

IonChannelProfiler ServiceAccess a range of ion channel assays—based on automated

and manual patch clamp electrophysiology—that support

different types of screening cascades, from HTS through

lead optimization and SAR studies. Whether it’s a 50,000

compound screen against an individual target, profiling

compound sets against a selectivity panel or a detailed

biophysical study of a single compound, EMD Millipore has the

expertise and technology to rapidly deliver the high quality

data you need.

Ion Channel Profiling ServicesIon channels are well known for regulating electrical activity

in excitable cells, and many roles in non-excitable tissues

continue to be uncovered. They are important therapeutic

targets in a range of indications including arrhythmia,

hypertension, local anesthesia, pain, stroke, epilepsy,

depression, bipolar disorder, COPD, autoimmune disorders

and diabetes.

In vitro hERG Profiling Service Our ion channel profiling technologies provide cutting-

edge, in vitro testing of compounds for hERG channel

blockage. Using robust cell lines generated in-house, we

have developed and validated high-quality functional assays

for reliably detecting hERG block in manual patch clamp,

PatchXpress® and IonWorks® systems.

IonWorks hERG assayCorrelation between IonWorks IC

50 values and published values obtained

using manual patch clamp for seven reference compounds. Most fall on the line of equivalence (shown in black), or within a 3-fold range of this (red dotted lines), indicating good correlation between methodologies.

Ion channels involved in mediating the cardiac action potential.

36

GPCR Profiling ServicesThere are ~385 druggable GPCRs (all non-odorant, non-

taste receptors), which share similar binding pockets.

Consequently, one drug may interact with more than one

receptor. Profiling a drug candidate’s GPCR activity can

reveal off-target effects, which can be either good or bad

for drug safety and efficacy.

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 ChemiScreenTM

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.

GPCR Profiling Reveals Interesting Off-target Hits for GPCR and Non-GPCR Directed Compounds.Clozapine, a marketed atypical antipsychotic, and BIRB0796, a compound developed as a SAPK2a/2b kinase inhibitor, were profiled at 10μM for agonist and antagonist activity against a large GPCR panel. Agonist activity was not detected for either compound. Percent inhibition is shown for the antagonist screen with the dotted line indicating 50% inhibition. BIRB0796 was not tested against CXCR6, MC4 and UT. Clozapine was not tested against BB3, CCR3, CX3CR1, XCR1, CCK1, ETB, GPR41, GAL2, GIP, GLP-1, secretin receptor, S1P1, MC2, GPR7, Y4, GPR109A, Mu, GPR103, PK2, EP2, IP1, sst5, GPR68 and NK.

AllostericProfiler ServiceAllostericProfiler uses a functional readout to detect

allosteric compound activity. AllostericProfiler is the

first fully validated selectivity-profiling service capable

of detecting a range of compound activities for over 155

GPCRs by using a unique two addition methodology to

detect a wide variety of activities including agonist, Positive

Allosteric Modulator (PAM) and Negative Allosteric Modulator

(NAM) activity. With the addition of AllostericScreenerTM

to our FlexLab capabilities, we can also be your partner

in identifying new positive allosteric modulators for your

favorite GPCR.

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KinaseProfiler™ and PhosphataseProfiler ServicePhosphate groups are key posttranslational modifications

to proteins in multiple toxicity pathways. Therefore, the

responsible modifying enzymes (kinases and phosphatases)

have been a focus of safety and liability testing.

KinaseProfiler and PhosphataseProfiler panels include

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 and phosphatase profiling

and screening, we provide validated data using the robust

and reliable radiometric kinase assay trusted by the world’s

leading pharmaceutical companies. With expertise to develop

hundreds of robust assays and consistent enzyme purity, we

provide experience, quality and proven excellence.

IC50Profiler™ ServiceOur IC50Profiler service enables you to follow up hits

identified in a standard KinaseProfiler study by determining

an IC50 value for your test compound against the kinase of

interest. Your report will include a graphical representation

of the data and the estimated IC50 value.

FlexLabSM ServicesIon Channel and GPCR FlexLab ServicesYour drug discovery program’s potential is not limited to our

existing products and services. Our FlexLab provides you with

experience, expertise and the flexibility you require to expand

your screening and profiling capacity where you need it. We

offer custom protein production for kinases, phosphatases

and protein substrates and custom cell line development

for GPCRs and ion channels. In addition, our custom assay

development team can design unique assays or extend our

current services to fit individual needs.

Dose Response of K252a-induced Neurite Outgrowth Inhibition.PC12 cells were cultured in low serum differentiation media containing 100 ng/mL NGF for 6 days, replacing media/NGF every 3 days. Cells received treatment with serial dilutions of the protein kinase inhibitor K252a, for the final 3 days of culture (max. concentration = 1000 nM). Cells were imaged on the GE IN Cell Analyzer 1000 (3.3) at 10X (10 fields/well) and analyzed using the GE IN Cell Analyzer 1000 Workstation 3.4) Neurite Outgrowth algorithm. (Mean ± SEM, n = 4).

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. Cells were imaged on the GE IN Cell Analyzer 1000. Shown: Fused images of Hoechst HCS nuclear stain (blue), phospho-histone H2A.X (green) and p53 (red) fluorescence.

Quantitative Cell Imaging (QCI) FlexLab ServicesQuantitative Cell Imaging (QCI) provides a means to extract

more information from cellular assays than ever before, via

automated image acquisition and quantitative image analysis.

EMD Millipore is developing innovative QCI applications for

drug discovery and safety testing, with a large and growing

portfolio of highly-validated assays that harness this exciting

technology, including:

• Neurite outgrowth

• Neurotoxicity

• Cell Cycle

• DNA Damage

• Cell Signaling

• Cellular Stress

• Hepatotoxicity

• QCI Assay Development

38

Bulk and Custom Services for Calbiochem and Novagen ProductsThe Custom Services Group (Calbiochem and Novagen brands)

welcomes partnerships with life sciences, pharmaceutical

and diagnostic organizations to provide innovative custom

solutions for research, distribution and manufacturing

needs. EMD Millipore manufacturing centers and dedicated

scientific and operations staff are ready to develop custom

formulations, alternate specifications and flexible packaging,

while meeting your strict quality assurance requirements and

ensuring lot-to-lot consistency. We also offer standing orders

and Just-In-Time delivery to help you manage your inventory.

Key Services Include:

o Assay Development - custom antibodies and inhibitors

o Cell Culture - competent cells and vectors

o Purification - detergents, separation assays and digestion

products

BioPharma ServicesThe World’s Large Molecule LabBiotherapeutics are changing the landscape of drug

development, creating a new paradigm and a new approach.

Scientists need a focused understanding of the development

of biologic therapies to advance their work. The shift

from small molecule research to large—coupled with new

regulatory requirements—has changed the face of how we

create the innovative compounds that treat disease and

improve human health. That’s why our BioPharma Services

Division has formed the world’s first global CRO dedicated

to large molecule bioanalytical work. Nowhere else can

scientists find the global lab resources and large molecule

expertise to handle any challenge, any size, any time,

anywhere.

Key Services Include:

o TK/PK Services

o PK Assessments of Biopharmaceuticals & Data Services

o Immunogenicity Services

o cGMP Services

o Biomarker Services

BioPharmaServices

GLPCompliance

Ligand BindingAssay Developmentand Validation

Drug Development

39

Scepter™ Handheld Automated Cell CounterCount cells and monitor toxicity with the first and only device to allow you to track your cell populations right at the culture hood.

Understanding your cells was never easier. The Scepter

cell counter (Cat. No. PHCC00000) detects and measures

the size of your cells, and displays the population as a

histogram of cell size distributions. From the histogram,

count all the cells or use the easy gating function to count a

chosen subpopulation. Monitor histograms over time or after

treatments for a quick and easy assessment of your cell

population’s health.

Use the Scepter cell counter to assess cell size changes caused by treatment with cytotoxic compounds.

600

500

400

300

200

100

0

A

C

6 8 10 12 14 16 18 20 22 24 26 28 30

Diameter

Cou

nt

B

D E

= Untreated: 95% viable. 5% dead/debris

= 50 µM Camptothecin 68% viable, 32% dead/debris

A: 6 – 28.66 μm: total cell population

B: 6 – 10.9 μm: debris & non-viable control 3T3

C: 10.9 – 28.66 μm: viable control 3T3

D: 6 – 12.51 μm: debris & non-viable induced 3T3

E: 12.51 – 28.66 μm: viable control 3T3

NIH 3T3 cells were treated and untreated with camptothecin, and counted using a Scepter cell counter. Histograms were generated and the different peaks were gated as indicated below. Scepter accurately assesses viable vs. non-viable populations as verified by flow cytometry.

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illipore.com/toxicity

Your Source for Toxicity ResearchEMD Millipore shares our customers’ goal of accelerating the delivery of safe and efficacious therapeutics to patients. Together with our customers, we continue to innovate breakthrough technologies and provide biologically relevant products towards achieving functional, high-throughput testing that predicts toxicity with a high level of accuracy.

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

TO PLACE AN ORDER OR RECEIVE TECHNICAL ASSISTANCE In the U.S. and Canada, call toll-free 1 800-Millipore (1-800-645-5476)

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

For more information or to order Calbiochem CompoundsOrders: 800 854 3417

Technical Support: 800 628 8470

E-mail: biosciencehelp@emdchemicals.com

Millipore, Advancing Life Science Together, MILLIPLEX, PrecisION, IHC Select, Guava, ViaCount and GPCRProfiler are registered trademarks of Millipore Corporation.The M mark, AXIS, Milli-Mark, MAPmate, FlowCellect, OxyICC, OxyIHC, OxyELISA, ToxReporter, Scepter, KinaseProfiler, AllostericProfiler, AllostericScreener, CardiacProfiler, IonChannelProfiler and IC50Profiler are trademarks of Millipore Corporation. FlexLab is a servicemark of Millipore Corporation.Calbiochem and Novagen are registered trademarks of EMD Chemicals.Luminex and xMAP are registered trademarks of Luminex Corporation.LumiGlo is a registered trademark of Kirkegaard & Perry Laboratories, Inc..EMD Millipore is a trademark of Merck KGaA.Fluoro-Jade and Fluoro-Ruby are registered trademarks of Histo-Chem, Inc.BLOTS is a trademark of GleneLinx International, Inc.Lit. No. PB3511ENUS Printed in U.S.A. 09/10 Rose© 2010 Millipore Corporation, Billerica, MA 01821 U.S.A. All rights reserved.

www.millipore.com/toxicity

Sublethal neurotoxicity in an in vitro neuronal cell system.Paclitaxel is a mitotic inhibitor used in cancer chemotherapy, however its use is associated with a toxic peripheral neuropathy. The image shows NGF-differentiated PC12 cells following 24 hr exposure to 1 μM paclitaxel. Cells were stained using reagents from catalog number HCS226, a neurotoxicity assay for Quantitative Cell Imaging [blue = Hoechst nuclei, green = neuronal βIII-tubulin, red = synaptophysin]. The cells in the image remain viable, however neurite length and number and synaptic puncta have been dramatically reduced by exposure to paclitaxel. Cells were imaged at 10X magnification using an IN Cell Analyzer HCA platform.

Image provided by Andrew Ball, PhD, Senior Research Scientist, EMD Millipore.

CELL STAINING IMAGE