Data SheetData Sheet

When it comes to drug development, today’s researchers

face tremendous challenges. The ability to know

everything you can about your drug compounds early

in the development process is critical to help avoid

unexpected, adverse effects during clinical trials. Drugs

for a wide variety of neurological diseases need to be

screened for toxicity during development.

Fortunately, Millipore’s new High Content Analysis (HCA) Neurotoxicity Assays overcome these challenges. By enabling simultaneous, rapid screening of multiple parameters in individual cells, you’re able to:• Run more tests in a shorter period of time

• Screen compounds for a broad range of potentially toxic

effects early in the drug discovery process

• Manage your lead compound projects with

increased efficiency

Leveraging the power of high-content screening, Millipore’s

HCA Neurotoxicity kits offer enhanced sensitivity, neuronal

specificity and the capability to detect multiple modes

of neuronal damage. As a result, these assays hold great

potential for advancing current neuroscience research,

including unraveling the mysteries of Alzheimer’s and

Parkinson’s diseases, and other neurodegenerative

disorders.

Millipore’s HCA Neurotoxicity Assays include assessment of cell number, neuronal- and glial-specific markers, such as: • Neurite outgrowth

• Synaptic vesicle formation

• Glial fibrillary acidic protein (GFAP) upregulation

• Astrocyte hypertrophy

At Millipore, we’re committed to providing you with the

best tools to do your best work, so you can get to the

accurate, reliable and marketable results you seek faster.

High Content Analysis:Neurotoxicity Assays

OrDeriNg iNFOrMAtiONDescription Catalogue No.

Neurotoxicity and Neurite Outgrowth Assay for High Content Screening HCS220

NeuroTox-1 Astrocyte Toxicity Assay for High Content Screening HCS221

NeuroTox-2 Astrocyte and Neuronal Toxicity Assay for High Content Screening HCS222

NeuroTox-3 Neuronal and Synaptic Toxicity Assay for High Content Screening HCS226

Nerve Growth Factor-b GF028

relAteD prODuCtSDescription Catalogue No.

Rat Hippocampal Neuron Kit SCR009

ReNcell™ CX Immortalized Cell Kit SCC009

ReNcell CX Immortalized Cell Line SCC007

ReNcell VM Immortalized Cell LIne SCC008

ReNcell CX Immortalized Cell Kit SCC009

ReNcell VM Immortalized Cell Kit SCC010

Cryopreserved Rat Hippocampal Astrocytes SCR008

Rat Hippocampal Neurons, 1x106 SCR010

Adult Rat Hippocampal Neural Stem Cells SCR022

Cryopreserved Mouse Cortical Neural Stem Cells SCR029

Rat Hippocampal Neurons, 1x106 SCR010

Cryopreserved Mouse Spinal Cord Neural Stem Cells SCR031

www.millipore.com/offices

tO plACe AN OrDer Or reCeiVe teCHNiCAl ASSiStANCe In the U.S. and Canada, call toll-free 1-800-MILLIPORE (1-800-645-5476)

Outside of North America, please visit www.millipore.com/offices

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

FOr MOre iNFOrMAtiON ON HigH CONteNt ANAlySiS prODuCtS Visit www.millipore.com/hca. For other ways Millipore supports

Life Science research, contact your local Millipore Drug Discovery

Specialist.

Neurotoxicity can result from exposure to a variety of agents, such as chemotherapeutic drugs, heavy metals, pesticides and solvents. 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.

THE EXPERTISE OF UPSTATE® AND CHEMICON®

IS NOW A PART OF MILLIPORE

NeurOtOx-3: NeurONAl AND SyNAptiC tOxiCity ASSAyThis kit enables evaluation of neuronal & synaptic

toxicity using HCA. The kit is also useful for

neurogenesis and synaptogenesis in neuronal

development. Traditional assays include estimating

neurite numbers and lengths by manual tracing of

neurons in culture, synaptic markers analyzed by

western blot or non-specific proliferation assays.

60,000

010-1310-1410-15 10-12 10-11 10-10 10-9 10-8 10-7 10-6 10-5

K252a (M, on log scale)

Neu

rite

Len

gth

(µm

, 10

fie

ld t

otal

)

80,000

100,000

AC50=1.03E-008

40,000

20,000

010-1310-1410-15 10-12 10-11 10-10 10-9 10-8 10-7 10-6 10-5

K252a (M, on log scale)

Syn

apto

phys

in (+

) A

rea

(µm

2, a

vg/c

ell)

80

0

Amyloid β (M, on log scale)

Syn

apto

phys

in (+

) A

rea

(µm

2, a

vg/c

ell)

60

40

20

140

120

100

80

60

40

20

140

120

100

50,000

010-13 10-12 10-11 10-10 10-9 10-8 10-7 10-6 10-5

Amyloid β (M, on log scale)

Neu

rite

Len

gth

(µm

, 10

fie

ld t

otal

)

10-4

10-13 10-12 10-11 10-10 10-9 10-8 10-7 10-6 10-5 10-4

70,00080,000

60,000

40,000

20,000

90,000

30,000

10,000

60,000

010-1310-1410-15 10-12 10-11 10-10 10-9 10-8 10-7 10-6 10-5

K252a (M, on log scale)

Neu

rite

Len

gth

(µm

, 10

fie

ld t

otal

)

80,000

100,000

AC50=1.03E-008

40,000

20,000

010-1310-1410-15 10-12 10-11 10-10 10-9 10-8 10-7 10-6 10-5

K252a (M, on log scale)

Syn

apto

phys

in (+

) A

rea

(µm

2, a

vg/c

ell)

80

0

Amyloid β (M, on log scale)

Syn

apto

phys

in (+

) A

rea

(µm

2, a

vg/c

ell)

60

40

20

140

120

100

80

60

40

20

140

120

100

50,000

010-13 10-12 10-11 10-10 10-9 10-8 10-7 10-6 10-5

Amyloid β (M, on log scale)

Neu

rite

Len

gth

(µm

, 10

fie

ld t

otal

)

10-4

10-13 10-12 10-11 10-10 10-9 10-8 10-7 10-6 10-5 10-4

70,00080,000

60,000

40,000

20,000

90,000

30,000

10,000

60,000

010-1310-1410-15 10-12 10-11 10-10 10-9 10-8 10-7 10-6 10-5

K252a (M, on log scale)

Neu

rite

Len

gth

(µm

, 10

fie

ld t

otal

)

80,000

100,000

AC50=1.03E-008

40,000

20,000

010-1310-1410-15 10-12 10-11 10-10 10-9 10-8 10-7 10-6 10-5

K252a (M, on log scale)

Syn

apto

phys

in (+

) A

rea

(µm

2, a

vg/c

ell)

80

0

Amyloid β (M, on log scale)

Syn

apto

phys

in (+

) A

rea

(µm

2, a

vg/c

ell)

60

40

20

140

120

100

80

60

40

20

140

120

100

50,000

010-13 10-12 10-11 10-10 10-9 10-8 10-7 10-6 10-5

Amyloid β (M, on log scale)

Neu

rite

Len

gth

(µm

, 10

fie

ld t

otal

)

10-4

10-13 10-12 10-11 10-10 10-9 10-8 10-7 10-6 10-5 10-4

70,00080,000

60,000

40,000

20,000

90,000

30,000

10,000

60,000

010-1310-1410-15 10-12 10-11 10-10 10-9 10-8 10-7 10-6 10-5

K252a (M, on log scale)

Neu

rite

Len

gth

(µm

, 10

fie

ld t

otal

)

80,000

100,000

AC50=1.03E-008

40,000

20,000

010-1310-1410-15 10-12 10-11 10-10 10-9 10-8 10-7 10-6 10-5

K252a (M, on log scale)

Syn

apto

phys

in (+

) A

rea

(µm

2, a

vg/c

ell)

80

0

Amyloid β (M, on log scale)

Syn

apto

phys

in (+

) A

rea

(µm

2, a

vg/c

ell)

60

40

20

140

120

100

80

60

40

20

140

120

100

50,000

010-13 10-12 10-11 10-10 10-9 10-8 10-7 10-6 10-5

Amyloid β (M, on log scale)

Neu

rite

Len

gth

(µm

, 10

fie

ld t

otal

)

10-4

10-13 10-12 10-11 10-10 10-9 10-8 10-7 10-6 10-5 10-4

70,00080,000

60,000

40,000

20,000

90,000

30,000

10,000

Dose responses to neurotoxic compoundsPC12 cells were differentiated in low serum/NGF for 6 days. Primary rat hippocampal astrocytes were cultured for 6 days prior to neuron addition. Cultures were treated with neurotoxins, then fixed and stained for combinations of bIII-tubulin, synaptophysin or GFAP. The 20X merged images above display PC12s stained for nuclei (Hoechst, blue), bIII-tubulin (green) and synaptophysin (red). Dose responses in neurite length (normalized to cell number), compared between mono-cultures of primary neurons and neuron/astrocyte co-cultures, displayed AC50 shifts for some toxins, potentially due to co-culture-generated neuroprotective effects.

pC12 cells differentiated with NgF for 6 daysStaining shows cellular responses after treatment with neurotoxins. Neurons are stained with nuclie (blue), bIII tubulin (green) and synaptophysin (red)

Panel A: DMSO control

Panel B: 0.1uM K252a (cells treated for 96h) Panel C: 10 uM amyloid beta (cells treated for 72h).

Images were acquired and analyzed using the GE Healthcare IN Cell 1000 Analyzer system.

A.

B.

C.

Millipore, Chemicon, and Upstate are registered trademarks of Millipore Corporation. The M mark and Advancing Life Science Together are trademarks of Millipore Corporation. FSC is a registered trademark of Forest Stewardship Council AC.ReNcell is a trademark of ReNeuron Limited Corporation.Lit. No. DS1256EN00 08DD026 11/08 Printed in U.S.A. DD-SBU-08-01205 © 2008 Millipore Corporation, Billerica, MA 01821 U.S.A. All rights reserved.

• Validated antibodies and dyes for high content analysis

that work on all HCA systems

• Fluorescence-based antibody assays that work on any

fluorescence or confocal microscope

• Multiplexed kits with specific control toxins and

compounds included, enabling you to gain functional data

on test compounds

• Validated protocols and recommended cell lines that

require no further assay development

Millipore’s HCA Kits include:• 96-well format

• Primary unconjugated and secondary conjugated antibodies

• All wash buffers, reagents, fixation solutions and plate sealers

• Control toxins

• 12-point dose response curve protocol

• Validated on multiple cell types

Achieve Better Detection and Assessment of Neurotoxins

NeurOtOx-2: NeurONAl AND AStrOCyte CO-Culture tOxiCity ASSAyThis kit has been shown to be more sensitive for

detection of low concentrations of neurotoxins than

traditional assays such as MTT and LDH tests. The

assay detects a combination of GFAP and neurite

outgrowth markers to evaluate toxicity in a co-culture

assay. The co-culture assay may provide a better in vitro

model than mono-culture.

Comparison of HCS neurite outgrowth analysis with Mtt and lDH assaysRat PC12 cells were cultured under differentiation conditions and treated with MPP+ and nocodazole (24h). Cells were fixed, stained and analyzed for total neurite length. Parallel cultures were analyzed utilizing MTT (colorimetric, signal proportional to live cell number) or LDH (colorimetric, signal proportional to dead cell number) assay kits, according to manufacturer’s instructions. Nocodazole-induced neuronal damage was not well detected by MTT or LDH, but HCS neurite outgrowth analysis displayed a dose-dependent effect.

Co-culture of astrocytes and neuronsRat PC12 cells were differentiated in low serum/NGF for 6 days. For co-culture, primary rat hippocampal astrocytes were cultured for 6 days prior to neuron addition. Cultures were treated with serial dilutions of neurotoxins, then fixed and stained for combinations of bIII-tubulin (green), and GFAP (red) as per protocol (Catalogue No. HCS222). Cells were imaged with a GE Healthcare IN Cell 1000 Analyzer. A= control, treated with H

2O.

B=10 mM Acrylamide (24h), C=1mM H

2O

2 (24h).

100,000

010-11 10-10 10-9 10-8 10-7 10-6 10-5 10-4 10-3 10-2 10-1

10-11 10-10 10-9 10-8 10-7 10-6 10-5 10-4 10-3 10-2 10-1

MPP+ (M, on log scale)

Neurite Length

MPP+: Comparison of Neurite Outgrowth Analysis with MTT Assay

Nacodazole: Comparison of Neurite Outgrowth Analysis with MTT Assay

MPP+: Comparison of Neurite Outgrowth Analysis with LDH Assay

Nacodazole: Comparison of Neurite Outgrowth Analysis with LDH Assay

MTT0.0003774EC50 0.0003466

Neu

rite

Len

gth

(µm

)

MTT (A

57

0)

120,000

Neurite Length (µm)

MTT (A570)

80,000

60,000

40,000

20,000

0.4

0.5

0.3

0.2

0.1

0.0

100,000

010-11 10-10 10-9 10-8 10-7 10-6 10-5 10-410-14 10-13 10-12

Nocodazole (M, on log scale)

Neurite Length MTT2.130e-007EC50 8.424e-012

Neu

rite

Len

gth

(µm

)

MTT (A

57

0)

120,000

Neurite Length (µm)

MTT (A570)

80,000

60,000

40,000

20,000

0.4

0.5

0.3

0.2

0.1

0.0

100,000

010-11 10-10 10-9 10-8 10-7 10-6 10-5 10-410-14 10-13 10-12

Nocodazole (M, on log scale)

Neurite Length2.130e-007EC50 5.027e-008

Neu

rite

Len

gth

(µm

)

LDH

(A4

90

)

120,000

Neurite Length (µm)

LDH (A490)

80,000

60,000

40,000

20,000

1.0

1.2

0.8

0.6

0.4

0.0

0.2

100,000

0

MPP+ (M, on log scale)

Neurite Length0.0003774EC50 0.0001329

Neu

rite

Len

gth

(µm

)

LDH

(A4

90

)

120,000

Neurite Length (µm)

LDH (A490)

80,000

60,000

40,000

20,000

1.2

1.0

0.8

0.6

0.2

0.0

0.4

LDH

LDH

100,000

010-11 10-10 10-9 10-8 10-7 10-6 10-5 10-4 10-3 10-2 10-1

10-11 10-10 10-9 10-8 10-7 10-6 10-5 10-4 10-3 10-2 10-1

MPP+ (M, on log scale)

Neurite Length

MPP+: Comparison of Neurite Outgrowth Analysis with MTT Assay

Nacodazole: Comparison of Neurite Outgrowth Analysis with MTT Assay

MPP+: Comparison of Neurite Outgrowth Analysis with LDH Assay

Nacodazole: Comparison of Neurite Outgrowth Analysis with LDH Assay

MTT0.0003774EC50 0.0003466

Neu

rite

Len

gth

(µm

)

MTT (A

57

0)

120,000

Neurite Length (µm)

MTT (A570)

80,000

60,000

40,000

20,000

0.4

0.5

0.3

0.2

0.1

0.0

100,000

010-11 10-10 10-9 10-8 10-7 10-6 10-5 10-410-14 10-13 10-12

Nocodazole (M, on log scale)

Neurite Length MTT2.130e-007EC50 8.424e-012

Neu

rite

Len

gth

(µm

)

MTT (A

57

0)

120,000

Neurite Length (µm)

MTT (A570)

80,000

60,000

40,000

20,000

0.4

0.5

0.3

0.2

0.1

0.0

100,000

010-11 10-10 10-9 10-8 10-7 10-6 10-5 10-410-14 10-13 10-12

Nocodazole (M, on log scale)

Neurite Length2.130e-007EC50 5.027e-008

Neu

rite

Len

gth

(µm

)

LDH

(A4

90

)

120,000

Neurite Length (µm)

LDH (A490)

80,000

60,000

40,000

20,000

1.0

1.2

0.8

0.6

0.4

0.0

0.2

100,000

0

MPP+ (M, on log scale)

Neurite Length0.0003774EC50 0.0001329

Neu

rite

Len

gth

(µm

)

LDH

(A4

90

)

120,000

Neurite Length (µm)

LDH (A490)

80,000

60,000

40,000

20,000

1.2

1.0

0.8

0.6

0.2

0.0

0.4

LDH

LDH

100,000

010-11 10-10 10-9 10-8 10-7 10-6 10-5 10-4 10-3 10-2 10-1

10-11 10-10 10-9 10-8 10-7 10-6 10-5 10-4 10-3 10-2 10-1

MPP+ (M, on log scale)

Neurite Length

MPP+: Comparison of Neurite Outgrowth Analysis with MTT Assay

Nacodazole: Comparison of Neurite Outgrowth Analysis with MTT Assay

MPP+: Comparison of Neurite Outgrowth Analysis with LDH Assay

Nacodazole: Comparison of Neurite Outgrowth Analysis with LDH Assay

MTT0.0003774EC50 0.0003466

Neu

rite

Len

gth

(µm

)

MTT (A

57

0)

120,000

Neurite Length (µm)

MTT (A570)

80,000

60,000

40,000

20,000

0.4

0.5

0.3

0.2

0.1

0.0

100,000

010-11 10-10 10-9 10-8 10-7 10-6 10-5 10-410-14 10-13 10-12

Nocodazole (M, on log scale)

Neurite Length MTT2.130e-007EC50 8.424e-012

Neu

rite

Len

gth

(µm

)

MTT (A

57

0)

120,000

Neurite Length (µm)

MTT (A570)

80,000

60,000

40,000

20,000

0.4

0.5

0.3

0.2

0.1

0.0

100,000

010-11 10-10 10-9 10-8 10-7 10-6 10-5 10-410-14 10-13 10-12

Nocodazole (M, on log scale)

Neurite Length2.130e-007EC50 5.027e-008

Neu

rite

Len

gth

(µm

)

LDH

(A4

90

)

120,000

Neurite Length (µm)

LDH (A490)

80,000

60,000

40,000

20,000

1.0

1.2

0.8

0.6

0.4

0.0

0.2

100,000

0

MPP+ (M, on log scale)

Neurite Length0.0003774EC50 0.0001329

Neu

rite

Len

gth

(µm

)

LDH

(A4

90

)

120,000

Neurite Length (µm)

LDH (A490)

80,000

60,000

40,000

20,000

1.2

1.0

0.8

0.6

0.2

0.0

0.4

LDH

LDH

100,000

010-11 10-10 10-9 10-8 10-7 10-6 10-5 10-4 10-3 10-2 10-1

10-11 10-10 10-9 10-8 10-7 10-6 10-5 10-4 10-3 10-2 10-1

MPP+ (M, on log scale)

Neurite Length

MPP+: Comparison of Neurite Outgrowth Analysis with MTT Assay

Nacodazole: Comparison of Neurite Outgrowth Analysis with MTT Assay

MPP+: Comparison of Neurite Outgrowth Analysis with LDH Assay

Nacodazole: Comparison of Neurite Outgrowth Analysis with LDH Assay

MTT0.0003774EC50 0.0003466

Neu

rite

Len

gth

(µm

)

MTT (A

57

0)

120,000

Neurite Length (µm)

MTT (A570)

80,000

60,000

40,000

20,000

0.4

0.5

0.3

0.2

0.1

0.0

100,000

010-11 10-10 10-9 10-8 10-7 10-6 10-5 10-410-14 10-13 10-12

Nocodazole (M, on log scale)

Neurite Length MTT2.130e-007EC50 8.424e-012

Neu

rite

Len

gth

(µm

)

MTT (A

57

0)

120,000

Neurite Length (µm)

MTT (A570)

80,000

60,000

40,000

20,000

0.4

0.5

0.3

0.2

0.1

0.0

100,000

010-11 10-10 10-9 10-8 10-7 10-6 10-5 10-410-14 10-13 10-12

Nocodazole (M, on log scale)

Neurite Length2.130e-007EC50 5.027e-008

Neu

rite

Len

gth

(µm

)

LDH

(A4

90

)

120,000

Neurite Length (µm)

LDH (A490)

80,000

60,000

40,000

20,000

1.0

1.2

0.8

0.6

0.4

0.0

0.2

100,000

0

MPP+ (M, on log scale)

Neurite Length0.0003774EC50 0.0001329

Neu

rite

Len

gth

(µm

)

LDH

(A4

90

)

120,000

Neurite Length (µm)

LDH (A490)

80,000

60,000

40,000

20,000

1.2

1.0

0.8

0.6

0.2

0.0

0.4

LDH

LDH

A.

B.

C.

NeurOtOx-1: AStrOCyte tOxiCity ASSAyThis assay detects glial fibrillary acidic protein (GFAP), a

marker of glial cells in the brain. Increases in GFAP are

considered to be evidence that a neurotoxic effect has

occurred. Use of GFAP and related glial biomarkers in

neurotoxicity screens may provide molecular signatures

predictive of adverse effects on the nervous system.

“Reactive gliosis” – a universal neurotoxicity biomarker,

can be characterized by altered GFAP expression, glial

cell hypertrophy and increased glial cell proliferation,

which is ideal for HCS analysis.

160

14510-13 10-12 10-11 10-10 10-9 10-8 10-7 10-6 10-5 10-4

Paclitaxel (M, on log scale)

GFA

P In

tens

ity

165 AC50=1.338E-007

155

150

160

145

14010-13 10-12 10-11 10-10 10-9 10-8 10-7 10-6 10-5 10-4

Nocodazole (M, on log scale)

GFA

P In

tens

ity

165 AC50=8.473E-008

155

150

160

14510-13 10-12 10-11 10-10 10-9 10-8 10-7 10-6 10-5 10-4

Paclitaxel (M, on log scale)

GFA

P In

tens

ity

165 AC50=1.338E-007

155

150

160

145

14010-13 10-12 10-11 10-10 10-9 10-8 10-7 10-6 10-5 10-4

Nocodazole (M, on log scale)

GFA

P In

tens

ity

165 AC50=8.473E-008

155

150

Astrocyte toxicity AssayRat hippocampal astrocytes were cultured with toxins and stained with the standard protocol (Catalogue No. HCS221). Cells were fixed and imaged with a GE Healthcare IN Cell 1000 Analyzer. Increased levels of GFAP were detected in a dose-response curve for each toxin.