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ProductSheet Data Selection Guide

Toxicity
Antibodies, Kits, Assays and Services

THE EXPERTISE OF UPSTATE, CHEMICON & LINCO IS NOW A PART OF MILLIPORE

Platforms, Technologies and Services


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.

Antibodies and Immunoassays


EMD 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 Analysis


EMD 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 Systems


Flow 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 Assays


MILLIPLEX 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 Compounds
Calbiochem 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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Introduction
The Relevance of Toxicity Studies
Toxicity 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 organspecific 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

Table of Contents
NEUROTOXICITY
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
MILLIPLEX MAP Rat Kidney Toxicity Panel 1

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MILLIPLEX MAg Human Kidney Toxicity Panel 4 Anti-Lipocalin-2 (LCN2)

Advances in Toxicity Testing


Improvements 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.

CELL HEALTH
Flow Cytometry Kits and Instrumentation ToxReporter Cell Lines and ELISAs

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FEATURED PRODUCTS: Calbiochem Lipopolysaccharides and DNA and RNA Polymerase Inhibitors

OXIDATIVE STRESS

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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

www.millipore.com/toxicity

TOXICITY SERVICES
Lead Discovery Services Bulk and Custom Services BioPharma Services

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Neurotoxicity
Due 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 Millipores 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.
Panel 1
MILLIPLEX MAP Human Neurodegenerative Disease Panel 1 Standard Curves

Median Fluorescence Intensity (MFI)

100,000

10,000

1,000

x x I x x I
I

x I
I

x I x

x I x

x x I

100

x x I
0.01 0.1

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

Apolipoprotein A1 Apolipoprotein CIII Apolipoprotein E

x x I

Prealbumin Complement Factor H Complement C3

2 Macroglobulin

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 L or less of sample. Recommended dilution for serum and plasma is 1:40,000 and recommended dilution for CSF is 1:400.

Panel 2
MILLIPLEX MAP Human Neurodegenerative Disease Panel 2 Standard Curves
x I x I
1,000

Panel 3
MILLIPLEX MAP Human Neurodegenerative Disease Panel 3 Standard Curves
x x I

Median Fluorescence Intensity (MFI)

x I

x I x x x x

Median Fluorescence Intensity (MFI)

100,000

100,000

10,000

10,000

x I x x I x I x x

x x I
1,000

x x I

x I x I x I
0.01

100

100

x I x
0.1

x
1 10 100 Concentration (ng/mL) 1,000 10,000

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

10 0.001

CRP 1-Antitrypsin

PEDF

x I

MIP-4 Complement C4

BDNF sVCAM-1 slCAM-1

SAP

x x I

MPO Cathepsin D PDGF-AA

PDGF-AB/BB RANTES PAI-1 (total)

NCAM

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

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

Relative Differences Between Normal and Ad Samples: Plasma


100 80 60 40 20 0 C3 100 80 60 40 20 0 sICAM-1 200 150 100 50 0 C4

Relative Differences Between Normal and Ad Samples: CSF and Plasma


0.04 0.03 0.02 0.01 0.00 PDGF-AA 15 10 5 0

a m

CS

Ad

Co

P value=0.009

Co

Pl

as

P value=0.007

P value=0.005

P value=0.028

CS

Relative Differences Between Normal and Ad Samples: CSF


1500 1000 500 0 Con CSF Ad CSF APO A1 1500 1000 500 0 Con CSF Ad CSF Prealbumin 25 20 15 10 5 0 SAP

2000 1500 1000 50 0

A1AT

Ad
P value=0.018

Con Plasma

Ad Plasma

Con Plasma

Ad Plasma

Con Plasma

Ad Plasma

250 200 150 100 50 0

m a Ad

CS

CS

as

Ad

Pl

P value=0.038

P value=0.003

P value=0.021

P value=0.006

Co

Con CSF

Ad CSF

Co

Pl
P value=0.002

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.

as

m a

www.millipore.com/toxicity
5

ng/mL pNF-H

Phosphorylated Neurofilament (pNF-H) Sandwich ELISA Kit


(Catalogue No. NS170) A Sensitive Biomarker of Axonal Injury The 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.

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Blood pNF-H in G93A SOD1

30

20

10

0 60 70 80 90 100 110 120 130

Age in Days
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 Neurotoxicity


The 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 immunofluorescencebased, 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.

A.

Neurotoxic properties of K252a Rat PC12 cells were treated with 0.4% DMSO (panel A) or 1 M 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), III-tubulin (green) (from Cat. No. HCS220) and synaptophysin (red) (from Cat. No. HCS226).

B.

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.

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.

0.9 0.8 0.7 0.6

Timecourse Analysis
AXIS microfluidic design:

OD450

0.5 0.4 0.3 0.2 0.1 0 0h 1h 6h 24h 48h

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 m) using the Milli-Mark FluoroPan neuronal marker (MAB2300X) shown in green, versus DAPI (blue). 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).

CHAMBER

MICROGROOVES

www.millipore.com/toxicity
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FEATURED PRODUCTS

Calbiochem Cholinesterase and Amyloidogenesis Inhibitors


Cholinesterase Inhibitors Under 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, membranebound vesicles concentrated near the synaptic end. Following their release from the pre-synaptic end, ACh molecules activate cholinoreceptors on the postsynaptic 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 Alzheimers 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 Inhibitors Research 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.

Muscarinic Effects
Cardiovascular Vasodilation Reduced Cardiac Rate Reduced Force of Contraction Gastrointestinal Increased Peristalsis Enhanced Secretary Activity Sphincter Relaxation

CHOLINE ESTERS

Granular Secretions Increased Pancreatic Secretions Enhanced Salivary K and Water Secretion Increased Lacrimal Secretions Increased Adrenal Medullary Secretions Urinary Bladder Increased Ureteral Peristalsis Reduced Bladder Capacity

Nicotinic Effects
CNS Effects Stimulation of CNS Excitation of Respiration Autonomic Ganglia Excitation of Sympathetic and Parasympathetic Ganglia Neuromuscular Junctions Muscle Contraction

A (-amyloid peptide) is a major component of neuritic plaques and cerebrovascular amyloid deposits in the brains of patients with Alzheimers disease (AD). The cellular origin of amyloid precursor protein (APP) that gives rise to A is now well understood. Morphological evidence suggests that APP-immunoreactive neurites, often capped by A deposits are a major source of parenchymal amyloid. However, other cells, including astroglia, microglia and vascular cells, may contribute to the formation of A. Because a long-standing hypothesis posits that A deposits are neurotoxic and are causative factors in the development and progression of AD, development and use of inhibitors of A fibrillogenesis are pivotal to neurotoxicity research.

OTHER KEY NEUROTOXICITY PRODUCTS:


Antibodies AB5864 AB5611 MAB5328 AXIS AX45005 AX45010 AX50010 AX90010 AXIS Axon Isolation Device, 450 m AXIS Axon Isolation Device, 450 m AXIS Axon Isolation Device, 500 m AXIS Axon Isolation Device, 900 m Anti-Myelin Basic Protein Anti-Neuroketal Anti-RAGE Lysates CL102 12-144 Multiplex Kits HBDP-33K HNP-35K HPT-66K RPT86K RSH69K HNDG4-36K HND1MAG-39 MILLIPLEX MAP Human Brain-Derived Protein Panel MILLIPLEX MAP Human Neuropeptide Panel MILLIPLEX MAP Human Pituitary Panel MILLIPLEX MAP Rat Pituitary Panel MILLIPLEX MAP Rat Stress Hormone Panel MILLIPLEX MAP Human Neurodegenerative Panel 4 Available Nov., 2010 MILLIPLEX MAg Human Neurological Disorders Panel 1 Available Nov., 2010 Rat Brain Microsomal Preparation, rat brain

Calbiochem Inhibitors 30967 345670 385885 171581 171587 171588 233165 287840 344079 345834 554325 ELISAs EZHS40 EZHS42 EZHS-SET EZBRAIN40 EZBRAIN42 EZBRAIN-SET NS400 NS690 NS830 High Sensitivity Human Amyloid 40 High Sensitivity Human Amyloid 42 High Sensitivity Human Amyloid 40 and 42 ELISA Set Human Amyloid 40 Brain ELISA Human Amyloid 42 Brain ELISA Human Amyloid 40 and 42 Brain ELISA Set a-Synuclein ELISA Kit Amyloid Precursor Protein (APP) ELISA Available Oct., 2010 Glial Fibrillary Acidic Protein (GFAP) ELISA Available Oct., 2010 Diisopropylfluorophosphate Galanthamine, Hydrobromide ()-Huperzine A A40 Fibrillogenesis Inhibitor A42 Fibrillogenesis Inhibitor II A42 Fibrillogenesis Inhibitor III Clioquinol Diclofenac Sodium Flurbiprofen Genistein, Soybean Resveratrol

HND2MAG-39K MILLIPLEX MAg Human Neurological Disorders Panel 2 Available Nov., 2010 Quantitative Cell Imaging Kits HCS221 HCS222 HCS226 Tissue Stains AG325 AG335 Fluoro-Jade C stain for neural tissue degeneration Fluoro-Ruby stain for acute axonal degeneration High Content Analysis Kit for Gliosis High Content Analysis Kit for Co-Culture of Neurons and Astrocytes High Content Analysis Kit for Neurite Outgrowth and Synaptic Activity

www.millipore.com/toxicity
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Visit our website for all toxicity-related products and services.

Cardiotoxicity
Cardiovascular 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 Lines


The 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 validatedusing both conventional and automated electrophysiologyand 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--go-gorelated 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--go-go-related gene (hERG) encodes the potassium ion channel responsible for delayed rectifier potassium current (IKr). There is a demand for highthroughput 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.

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.

100,000

MILLIPLEX MAP Rat CVD Panel 1 Standard Curves

Median Fluorescence Intensity (MFI)

10,000

1,000

100

900

Ion Channel hERG Membrane

[3H]-Astemizole bound (cpm)

800 700 600 500 400 300 200 100 0 -10 -9 -8 -7 -6 -5 -4

10

00

10

10

00

1,

,0

10

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


BNP MCP-1 Tnl MPO IL-6 vWF TIMP-1 TnT TNF- PAI-1 (total) VEGF

[Compound] Log M

Astemizole Dofetilide

E-4031 Cisapride

Verapamil WT

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 L sample volume.

Rank ordering small molecule inhibitors for hERG. hERG Membrane Preparation (10 g/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.

Troponin T Normal vs. SHR Serum


400

pg/mL

350 300 250 200 150 100 50 0

Normal

SHR

10

0,

00

www.millipore.com/toxicity

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.

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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.

A.

Before After Centrifugation Centrifugation

B.

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.

FEATURED PRODUCTS

Calbiochem Ionophores and Calcium Channel Modulators


Ionophores Ionophores 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 Ca concentrations or
2+

Calcium Signaling Products, Including IP3, Ryanodine and Calcium Channel Modulators Toxicology-related pharmacological, electrophysiological and biochemical investigations include an evaluation of calcium channel modulation effects on excitationcontraction 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.

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.

12

OTHER KEY CARDIOTOXICITY PRODUCTS:


Antibodies AB1549 AB5412 06-382 MAB3458 06-811 05-205 07-052 AB5930 AB5932 MAB2636 MAB1693 MAB3150 MAB3152 Anti-Brain Natriuretic Peptide (BNP) Anti-Calcium Channel, Voltage Gated Cardiac a1C Anti-Calsequestrin, cardiac Anti-Myofibroblasts Anti-Na+ Channel a, cardiac (III-IV loop) Anti-Phospholamban, clone A1 Anti-phospho-Phospholamban (Ser16) Anti-Potassium Channel ERG1, C-terminus Anti-Potassium Channel KvLQT1, C-terminus Anti-SERCA2, clone IID8 Anti-Troponin T, clone 2G3 Anti-Cardiac Troponin I, a.a. 41-49, clone 284 (19C7) Anti-Cardiac Troponin I, a.a. 87-91, clone 8E10 SCM101 SCM102 RCVD2-89K RCVD3-89K HCVD2-67BK MCVD1-77AK MCVD277BK Lysates CL104 Rat Heart CL304-250UG Human Heart Multiplex Kits HCVD1-67AK MILLIPLEX MAP Human Cardiovascular Disease (CVD) Panel 1 MILLIPLEX MAP Human Cardiovascular Disease (CVD) Panel 2 MILLIPLEX MAP Mouse Cardiovascular Disease (CVD) Panel 1 MILLIPLEX MAP Mouse Cardiovascular Disease (CVD) Panel 2 MILLIPLEX MAP Rat Cardiovascular Disease (CVD) Panel 2 MILLIPLEX MAP Rat Cardiovascular Disease (CVD) Panel 3

Calbiochem Inhibitors & Modulators 115500 100065 286888 203675 251680 298711 682160 682162 512743 Adenophostin A, Hexasodium Salt 2-APB BHQ Bombesin, Free Base Dantrolene, Sodium Salt Diethyl Pyrocarbonate Xestospongin C, Xestospongia sp. Xestospongin D, Xestospongia sp. Pasteurella Multocida Toxin, Pasteurella multocida

Stem Cell Reagents Cardiac Stem Cell Maintenance Medium Cardiomyocyte Differentiation Medium

Calbiochem Ionophore Related Products 100107 100105 100106 205535 368020 407952 407950 475897 475895 481990 475914 569385 676377 A23187, 4 Bromo A23187, Free Acid, Streptomyces chartreusensis A23187, Mixed Calcium-Magnesium Salt CA 1001 Gramicidin A, High Purity, Bacillus brevis Ionomycin, Calcium Salt, Streptomyces conglobatus Ionomycin, Free Acid, Streptomyces conglobatus Monensin Methyl Ester Monensin, Sodium Salt, High Purity Nigericin, Sodium Salt, Streptomyces hygroscopicus Nystatin, Streptomyces noursei SQI-Pr Valinomycin, Streptomyces fulvissimus

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13

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Hepatotoxicity
Drug-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 druginduced 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.

A.

B.

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 phosphohistone H3 levels.

14

Median Fluorescence Intensity (MFI)

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.

MILLIPLEX
100,000 10,000 1,000 100 10 1 0.01

MAG

Human Liver Protein Panel Standard Curves

0.1

10

100

1,000

10,000

Concentration (ng/mL)

AFP ANGPTL3 ANGPTL4 ANGPTL6

HGF FABP1 FGF-19 FGF-21

FGF-23

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

140.00 120.00 100.00

ANGPTL4

1.40 1.20 1.00

HGF

18.00 16.00 14.00 12.00

FGF-23

pg/mL

pg/mL

pg/mL
Normal Disease

80.00 60.00 40.00 20.00 0.00 Normal Disease

0.80 0.60 0.40 0.20 0.00

10.00 8.00 6.00 4.00 2.00 0.00 Normal Disease

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.

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15

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-Cytochrome P450 CYP450 1A2


Western Blot Analysis: Baculovirus-expressed recombinant rat CYP450 1A2 was resolved by electrophoresis, 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 AB9916 AB10080 AB10088 MAB10111 AB10324 FCMAB115F MAB4349 MAB4354 MAB5324 AB10339 Anti-CYP2A6 Anti-CYP2b10 Anti-CYP450, clone 2E1 Anti-CYP450 1A1 Anti-CYP450 4F11, clone F21 P6 F5 Anti-CYP450 Pan 2C Anti-TRA-1-60, clone TRA-1-60 FITC conjugate Anti-TRA-2-49, Liver/Bone/Kidney Alkaline Phosphatase, clone TRA-2-49/6E Anti-TRA-2-54, Liver/Bone/Kidney Alkaline Phosphatase, clone TRA-2-54/2J Anti-Polysialic Acid-NCAM, clone 2-2B Anti-UGT1a1 Blots TB030 Multiplex Kits HLPP-57K MILLIPLEX MAP Human Liver Protein Panel Ready-to-Screen Tissue BLOTS Human Liver

Tissue protein extracts and lysates CL108 CL208 CL308 SCC001 Rat Liver Mouse Liver Human Liver Human Neonatal Liver Cell Suspensions

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16

Nephrotoxicity
Because 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, -2-Microglobulin, Clusterin, Cystatin C and Osteopontin (OPN). Most of these biomarkers are included in the list from the Critical Path Institutes Predictive Safety Testing Consortium (PSTC) and are considered qualified for particular uses in regulatory decision making for acute kidney injury (AKI).

MILLIPLEX
100,000

MAG

Human Kidney Toxicity Panel 4 Standard Curves

Median Fluorescence Intensity (MFI)

10,000

1,000

Clusterin 100 Albumin 2M Cystatin OPN (ON) 10

10

10

1,0

00

10

,00

10

0,0

00 1,0

00

,00

0
, 10 00

0,

00

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 L sample volume of a 1:50 dilution is required.

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17

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
140 120 100 3000 2500 2000

MAG

Human Kidney Toxicity Panel 4 in Urine Samples


14000 12000 10000
*

5000 4000

1400 1200 1000

g/mL

g/mL

g/mL

g/mL

80 60 40 20 0

8000 6000 4000 200


*

g/mL Cystatin C

3000 2000 1000 0

800 600 400 200 0

1500 1000 500 0

Albumin

B2M On Cisplatin

Clusterin

OPN

Normal

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.

MILLIPLEX map Rat Kidney Toxicity Panel 1


(Catalogue No. RKTX1-37K) The rat is commonly used as a model in kidney disease and nephrotoxicity studies. The MILLIPLEX MAP Rat Kidney 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).
Median Fluorescence Intensity (MFI)
100,000

MILLIPLEX

MAP

Human Kidney Toxicity Panel 1 Standard Curves

Toxicity Panel 1 was developed to aid scientists in the

10,000

1,000

100 Clusterin (2 hr) KIM-1 (2 hr) 10 OPN (2 hr) Clusterin (ON) KIM-1 (ON) OPN (ON) 1

10

10

1,0

00

10

,00

10

0,0

00

1,

00

0,

00

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 L neat urine sample volume required.

18

20,000

CLUSTERIN

400 350

KIM-1

600 500 400

OSTEOPONTIN

15,000

300 250

pg/mL

pg/mL

10,000

200 150

pg/mL
Vehicle Gentimicin NPA

300 200 100 0 Vehicle Gentimicin NPA

5,000

100 50

0 Vehicle Gentimicin NPA

Day 1

Day 4

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).

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.

40 35 30 25

1,400 1,200 1,000 800

160 140 120 100

ng/mL

pg/mL

20 15 10 5 0

pg/mL KIM 1 Wistar Males

80 60 40 20 0

600 400 200 0

CLUSTERIN Sprague Dawley Males

OSTEOPONTIN Wistar

Sprague Dawley Females

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.

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19

Anti-Lipocalin-2 (LCN2)
(Catalogue No. AB2267) LCN2 (also known as NGAL, or neutrophil gelatinaseassociated 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 Millipores anti-LCN2 antibody has been validated for immunohistochemistry or Western blotting.

OTHER KEY NEPHROTOXICITY PRODUCTS:


Antibodies 05-354 05-355 05-356 CBL209F CBL306 CBL307 CBL307-K AB10910 AB1870 MAB3055 MAB10212 ELISAs EZHRBP4-18K EZMAGP-23K Lysates 12-146 CL106 CL206 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 Microsomal Preparation, rat kidney Rat Kidney Mouse Kidney Human RBP4 ELISA Mouse AGP ELISA Anti-Clusterin a chain (human), clone 41D Anti-Clusterin chain (rat) Anti-Clusterin a chain (rat), clone 7A8-E4 Anti--2 Microglobulin, clone GJ14, FITC conjugated Anti--2 Microglobulin, clone C21 Anti--2 Microglobulin, clone B2 Anti--2 Microglobulin, Clone B2 (bulk size) Anti-Osteopontin (human, mouse) Anti-Osteopontin (human, mouse, rat) Anti-Osteopontin, recombinant protein only, clone 4AA Anti-Renin, clone F32 VIII C4

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).

HKTX3-38K

MILLIPLEX MAP Human Kidney Toxicity Panel 3

HKTX3MAG-38K MILLIPLEX MAg Human Kidney Toxicity Magnetic Bead Panel 3 HKTX4-38K RKTX2-37K MILLIPLEX MAP Human Kidney Toxicity Panel 4 MILLIPLEX MAP Rat Kidney Toxicity Panel 2

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20

Cell Health
Adverse 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 Instrumentation


The 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 L of sample.

Debris

Viable Cells

Apoptotic

Dead Cells

Forward Scatter Nuclear Stain Viability Stain

Low Neg Neg

High High Neg

High High Med

High High High

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

www.millipore.com/toxicity
21

Guava Cell Toxicity Assay


(Catalogue No. 4500-0230) Small molecules and other therapeutics often cause toxicity by stimulating the bodys 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. B.
Live Effector Live Target cells cells

Guava Cell Toxicity Assay A.


Dead Effector cells Dead Target cells

7-AAD

CFSE

How the Guava Cell Toxicity Assay Works


Target cell is labeled with cell tracking dye Target cell is killed by effector cell Membrane is damaged Dead target cell

Add dead cell dye: 7-AAD

(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.

Effector cell

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.

22

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.

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 AntiBrdU 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)

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

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. 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.

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23

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 (7AAD), 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.

104

Uninduced
94.4% 1.1%
Red2 Fluoresecence (RD2-HLog)

104

2 M Staurosporine
54.7% 3.7%
Red2 Fluoresecence (RD2-HLog)

104

50 M CCCP
0.20% 0.04%

Red2 Fluoresecence (RD2-HLog)

103

103

103

MitoSense Red

102

102

102

101

101

101

100 100

0.75%
101 102 103
Green Fluorescence (GRN-HLog)

3.7%
104

100 100

14.6%
101 102 103

27.0%
104

100 100

93.2%
101 102 103
Green Fluorescence (GRN-HLog)

6.6%
104

Green Fluorescence (GRN-HLog)

Annexin V, CF488A
104

95.2%

0.3%
Red2 Fluorescence (RD2-HLog)

104

58.1%

0.08%
Red2 Fluorescence (RD2-HLog)

104

0.26%

0.02%

Red2 Fluorescence (RD2-HLog)

Live Cells
103

103

103

MitoSense Red

102

102

Dead Cells

102

101

101

101

Depolarized Cells
100 100

3.2%
101 102 103
Red Fluorescence (RED-HLog)

1.3%
104

100 100

41.0%
101 102 103
Red Fluoresecence (RED-HLog)

0.8%
104

100 100

98.4%
101 102 103
Red Fluorescence (RED-HLog)

1.3%
104

104

0.16%

1.4%
Red2 Fluorescence (RD2-HLog)

104

7-AAD
0.06% 1.2%
Red2 Fluorescence (RD2-HLog)

104

0.10%

1.2%

Red2 Fluorescence (RD2-HLog)

103

103

103

102

102

102

7-AAD

101

101

101

100 100

95.2%
101 102 103
Green Fluorescence (GRN-HLog)

3.2%
104

100 100

70.5%
101 102

28.2%
103 104

100 100

93.9%
101 102 103
Green Fluorescence (GRN-HLog)

4.8%
104

Green Fluorescence (GRN-HLog)

Annexin V, CF488A

Dot plots depicting Jurkat cells stained using the MitoDamage kit. Jurkat cells uninduced (left column), induced to apoptosis with 2 M staurosporine (center column) or with 50 M 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 change 3rd row: Apoptosis and cell death Data reports that 2 M staurosporine induces apoptosis in Jurkat cells, and that 50 M CCCP depolarizes the mitochondrial membrane, but neither condition is sufficient for cell membrane permeabilization and death.

24

ToxReporter Cell Lines and ELISAs


An innovative platform to measure toxicity levels in a variety of pathways using a single ELISA
ToxReporter 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 Lines


Specific Response Element p21 AP-1 Catalogue Number, Cell Line ECL-001 ECL-002 Catalogue Number, Starter Pack* ECLSP-001 ECLSP-002 Potential Toxicity Mechanism DNA damage Oxidative stress, DNA damage and Apoptosis Immunosuppressant Cellular stress, Heavy metal toxicity Oxidative stress, DNA damage Comment p21 is associated with DNA damage. Activator Protein-1 is a redox sensitive transcription factor associated with oxidative stress, DNA damage and apoptosis. Effect is down regulated by activated GR (Glucocorticoid receptor). Glucocorticoid Response Element regulates immunosuppressive and anti-inflammatory activities in multiple physiological systems. Heat Shock Protein 70 is a chaperone protein associated with heat shock, metal toxicity and cellular stress responses. A redox sensitive transcription factor associated with oxidative stress, DNA damage and apoptosis. Effect is down regulated by activated GR (Glucocorticoid receptor). Hypoxia Response Element associated with hypoxic stress, DNA damage, especially mitochondrial. Xenobiotic Response Element is associated with the AR receptor (Aryl hydrocarbon Receptor) which induces the expression of cytochrome P450s (1A1). Anti-Oxidant Response Element leads to the expression of a number of phase II detoxification genes in response to electrophilic compounds or oxidants. Heme oxygenase 1 is associated with oxidative stress response. p53 Response Element is associated with DNA damage.

GRE

ECL-003

ECLSP-003

HSP70

ECL-004

ECLSP-004

NFKB

ECL-005

ECLSP-005

HRE XRE

ECL-006 ECL-007

ECLSP-006 ECLSP-007

Mitochondrial DNA damage Induction of CYP450s

ARE

ECL-008

ECLSP-008

Oxidative stress

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Hmox1 p53

ECL-009 ECL-010

ECLSP-009 ECLSP-010

Oxidative stress DNA damage

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

25

1 - 10 L Cultured Media

600

AP-1 Dose Response EC50 for TPA = 1.5 nM

ToxR ToxReporter ELISA

ToxReporter (pg/mL)

400

200

Potential Toxin

CELL

0 -14 -12 -10 -8 -6

Induction of Toxic Response Pathway

Transcription Factor

Log [TPA] M
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).

Responsive Promoter

Secreted ToxReporter

ToxReporter (pg/mL)

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.

AP-1 Time Course


800 600 400 200 0 -12 -11 -10 -9 -8 -7 -6

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.

Log [TPA] M
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 L 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.

26

FEATURED PRODUCTS

Calbiochem Lipopolysaccharides and DNA and RNA Polymerase Inhibitors


Lipopolysaccharides Lipopolysaccharides (LPS) act as the prototypical endotoxin because they bind the CD14/TLR4/MD2 receptor complex, which promotes the secretion of proinflammatory 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 Inhibitors DNA 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.

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OTHER KEY CELL HEALTH PRODUCTS:


Antibodies MAB4122 MAB4140 MAB4163 MAB448 CT01 CT02 2210 Anti-MDR related Protein, clone MRPm6 Anti-MDR3, clone P3 II-26 Anti-MDR1, clone MC57 Anti-MDR1, clone 3C3.2 MTT Cell Growth Assay Kit MTT Cell Growth Assay Kit Cell Proliferation Assay Kit, WST dye; ELISA based Guava ViaCount Related Products 4500-0110 4700-0050 MAPmates 46-665 46-607 46-608 46-613 46-618 46-610 46-612
TM

Guava ViaCount Flex Reagent Kit For Challenging Samples Guava ViaCount Cell Dispersant Reagent Kit

Total HIF1a MAPmate Phospho HSP27 (Ser78) MAPmate Total HSP27 MAPmate Phospho JNK/SAPK1 (Thr183/Tyr185) MAPmate Total JNK/SAPK1 MAPmate Phospho p38/SAPK (Thr180/Tyr182) MAPmate Total p38/SAPK MAPmate

Calbiochem Inhibitors 129935 178273 385883 491207 557403 Actinomycin D, 7-Amino Aphidicolin HSV Replication Inhibitor, BP5 Novobiocin, Sodium Salt RNA Polymerase III Inhibitor

Quantitative Cell Imaging Kits HCS210 HCS211 HCS212 HCS213 HCS216 HCS223 HCS224 HCS225 HCS231 HCS235 HCS236 HCS237 Cyclin B1 and Ki-67 assay Phospho-Histone H3 (Ser10) and Cyclin B1 Assay BrdU and Phospho-Histone H3 (Ser10) Assay Brdu and Ki-67 Assay Phospho-Histone H3 and a tubulin Assay p53/ DNA Damage Assay H2A.X Phosphorylation/DNA Damage Assay H2A.X Phosphorylation and p53 DNA Damage Assay p38 MAPK Assay Kit p53 and p21 Assay Kit Cytochrome C Assay c-Jun Activation Assay

Calbiochem Lipopolysaccharides 437620 437627 437625 437629 437650 437628 Lipopolysaccharide, E. coli J5 Lipopolysaccharide, E. coli O111: B4 Lipopolysaccharide, E. coli O55: B5 Lipopolysaccharide, Salmonella minnesota Re 595 Lipopolysaccharide, Salmonella typhimurium Lipopolysaccharide, Ultra Pure, Salmonella minnesota R595 (Re)

FlowCellect Kits FCCH025143 FCCH025142 FCCH100105 FCCH100107 FCCH100109 FCCH100110 FCCH025111 FlowCellect Cell Cycle CheckPoint ATM DNA Damage Kit FlowCellect Cell Cycle CheckPoint H2A.X DNA Damage Kit FlowCellect MitoPotential Red Kit FlowCellect MitoLive Kit FlowCellect MitoStress Kit FlowCellect Cytochrome C Kit FlowCellect Oxidative Stress Characterization Kit

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 overexercise. There is increasing evidence that free radicals damage biomolecules, leading to several specific and diverse diseases, such as atherosclerosis, cerebral and heart ischemiareperfusion injury, cancer, rheumatoid arthritis, inflammation, diabetes, aging and neurodegenerative conditions such as Alzheimers 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 Millipores 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 prooxidant insults resulting from cytokine treatment, ultraviolet light, irradiation, certain tumors, amyotrophic lateral sclerosis and ischemia/reperfusion.

Kit: Blue: Green: Red: Cells:

HCS233 Hoechst nuclear stain Phospho-Histone H2A.X (Ser139) Manganese Superoxide Dismutase (MnSOD) HeLa cervical adenocarcinoma

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Assays for the Detection of Oxidized Proteins


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

OxyICC Oxidized Protein Detection 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.

(H2O2, NO, Superoxides etc.) Protein sample from cell lysate, tissue homogenate, biological fluids.

Oxidative Stress

H2N NH O + NO2 NO2

DNPH

A.

B.

A: -H202 / -DNPH B: +H202 / -DNPH C: -H202 / +DNPH D: +H202 / +DNPH

C.
N NH NO2 DNP-derivatized protein

D.

NO2

+
Y Y
NO
2

Anti-DNP conjugated to HRP

Y
N NH

Immunodetection via WB, EIA, IC, IH, FC

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.

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NO

OxyIHC Oxidative Stress Detection 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.

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.

A.

B.

Arginine

NOS

Citrulline GTP

02

Phox

02-

NO

sGC

C.

D.
cGMP SOD 0N00Nitrotyrosine/ Protein

H202 + 02
The OxyIHC assay (Cat. No. S4750) identified oxidative stress in the cerebellar cortex of the Alzheimers 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 Alzheimers disease transgenic mice are under increased oxidative stress.

9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0

Nitrotyrosine ELISA

CPS x 106

www.millipore.com/toxicity

pe

tit o

0.1

10

100

1,000

10,000

No

Co

Nitrated BSA (g/mL)

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

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FEATURED PRODUCTS

Calbiochem Nitric Oxide Synthase, Arginase and Glutathione S-Transferase (GST) Inhibitors
Nitric Oxide Synthase (NOS) Inhibitors Low 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 Inhibitors Arginase 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 0N00-

DNA Breaks

Base Damage Apoptosis

O L-Arg NOS NO Cytostasis

Tumor Regression L-Cit Fe GTP


2+

GC

EC Proliferation/ Migration Anglogenesis Tumor Progression Blood Flow

cGMP cGMP-Gated Ion Channel cGMPPDE

Vasodilation PKG Activation

Glutathione S-Transferase (GST) Inhibitors Glutathione 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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OTHER KEY OXIDATIVE STRESS PRODUCTS:


Antibodies AB1284 AB5480 AB5830 AB9328 MAB3560 MAB5382 Anti-Heme Oxygenase1 Anti-SOD1 Anti-8-Hydroxydeoxyguanosine Anti-Thioredoxin 1 Anti-8-Oxoguanine Anti-HIF-1 a ELISA & Western Blot Kits S7150 S7250 OxyBlot Protein Oxidation Detection Kit OxyELISATM Oxidized Protein Quantitation Kit

Quantitative Cell Imaging HCS232 HCS234 Manganese Superoxide Dismutase (MnSOD) Assay p21 Detection Assay for High Content Screening

Calbiochem Inhibitors & Modulators 56766 288500 300260 311204 466220 483120 483125 490075 567300 691550 100050 154500 197900 205546 265005 311203 341180 400600 444600 472804 475886 482100 483400 490070 548000 589411 Spermidine, Trihydrochloride DL-a-Difluoromethylornithine, Hydrochloride Diphenyleneiodonium Chloride NG,NG-Dimethyl-L-arginine, Dihydrochloride S-Methylisothiourea, Sulfate NG-Nitro-L-arginine NG-Nitro-L-arginine Methyl Ester, Hydrochloride Nitric Oxide Synthase Inhibitor Set SKF-525A, Hydrochloride Zinc (II) Protoporphyrin IX 1400W Aminoguanidine, Hemisulfate BEC, Hydrochloride Caffeic Acid Dexamethasone NG,NG-Dimethyl-L-arginine, Dihydrochloride 2-Ethyl-2-thiopseudourea, Hydrobromide L-N5-(1-Iminoethyl)ornithine, Dihydrochloride MEG, Hydrochloride S-Methyl-L-thiocitrulline, Dihydrochloride NG-Monomethyl-L-arginine, Monoacetate Salt L-NIL, Dihydrochloride 7-Nitroindazole Nitric Oxide Synthase, Neuronal Inhibitor I 1-Pyrrolidinecarbodithioic Acid, Ammonium Salt L-Thiocitrulline, Dihydrochloride

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

Toxicity Services
Providing critical early assessment across a range of targets, EMD Millipores 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 candidatesincluding potential off-target liabilitiesyoull avoid costly errors and develop effective compounds faster.

Safety & Liability Screening Panel


Drug 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

hKir2.1 hKv4.3/hkChIP1

Ion Channel

hCav1.2 hCav1.2 hKCNQ1/hminK hHCN4 hKv1.5 hERG

20

40

60

80

100

% Inhibition

BIRB0796 Inhibition of Cardiac Ion Channels BIRB0796 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 M against cardiac ion channels included in the Safety & Liability Screening Panel. Results indicate significant inhibition of hERG and hKv1.5 currents, indicating potential cardiotoxicity.

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Ion Channel Profiling Services


Ion 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.

CardiacProfiler Service
CardiacProfiler 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.

In vitro hERG Profiling Service


Our ion channel profiling technologies provide cuttingedge, 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.

Ion channels involved in mediating the cardiac action potential.

IonChannelProfiler Service
Access a range of ion channel assaysbased on automated and manual patch clamp electrophysiologythat support different types of screening cascades, from HTS through lead optimization and SAR studies. Whether its a 50,000 compound screen against an individual target, profiling compound sets against a selectivity panel or a detailed
IonWorks hERG assay Correlation between IonWorks IC50 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.

biophysical study of a single compound, EMD Millipore has the expertise and technology to rapidly deliver the high quality data you need.

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GPCR Profiling Services


There are ~385 druggable GPCRs (all non-odorant, nontaste receptors), which share similar binding pockets. Consequently, one drug may interact with more than one receptor. Profiling a drug candidates GPCR activity can reveal off-target effects, which can be either good or bad for drug safety and efficacy.

AllostericProfiler Service
AllostericProfiler 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.

GPCRProfiler Service
GPCRProfiler 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 10M 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.

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KinaseProfiler and PhosphataseProfiler Service


Phosphate 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 worlds leading pharmaceutical companies. With expertise to develop hundreds of robust assays and consistent enzyme purity, we provide experience, quality and proven excellence.

Quantitative Cell Imaging (QCI) FlexLab Services


Quantitative 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

IC50Profiler Service
Our 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.

FlexLab Services
SM

Ion Channel and GPCR FlexLab Services


Your drug discovery programs 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).

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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.

Bulk and Custom Services for Calbiochem and Novagen Products


The 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.

BioPharma Services
The Worlds Large Molecule Lab
Biotherapeutics 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 largecoupled with new regulatory requirementshas changed the face of how we create the innovative compounds that treat disease and improve human health. Thats why our BioPharma Services Division has formed the worlds 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 Assay Development - custom antibodies and inhibitors o Cell Culture - competent cells and vectors o Purification - detergents, separation assays and digestion products

Key Services Include:


o TK/PK Services o PK Assessments of Biopharmaceuticals & Data Services o Immunogenicity Services o cGMP Services o Biomarker Services

GLP Compliance

Ligand Binding Assay Development and Validation

BioPharma Services

Drug Development

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Scepter Handheld Automated Cell Counter


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

600
A

500 400

Count

300 200 100 0 6


B C D E

10

12

14

16

18

20

22

24

26

28

30

Diameter

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

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

A: 6 28.66 m: total cell population B: 6 10.9 m: debris & non-viable control 3T3 C: 10.9 28.66 m: viable control 3T3 D: 6 12.51 m: debris & non-viable induced 3T3 E: 12.51 28.66 m: 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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Your Source for Toxicity Research


EMD 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
CELL STAINING IMAGE 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 M paclitaxel. Cells were stained using reagents from catalog number HCS226, a neurotoxicity assay for Quantitative Cell Imaging [blue = Hoechst nuclei, green = neuronal III-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.

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 Compounds


Orders: Technical Support: E-mail: 800 854 3417 800 628 8470 biosciencehelp@emdchemicals.com

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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.