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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.