Download Neuroscience - Thermo Fisher Scientific

Neuroscience
Publications List
2013 Edition
What is High Content Screening (HCS)?
High content screening (HCS), also known as high content analysis, image cytometry, quantitative cell analysis or
automated cell analysis, is an automated method that is used to identify substances that alter the phenotype of a
cell in a desired manner. This technology is primarily used in biological research and drug discovery and combines
fluorescent microscopy, automated cell calculations, and phenotyping using image processing algorithms and
informatics tools for the user to make decisions about a treatment.
Thermo Scientific High Content Products
The portfolio of High Content Analysis products includes assay development and screening tools like the Thermo
Scientific™ ArrayScan™ XTI High Content Analysis (HCA) Reader and the Thermo Scientific™ CellInsight™ NXT
High Content Screening (HCS) Platform. Multiple tools are available for assay development on the ArrayScan XTI
HCA Reader like the Thermo Scientific™ X1 large field-of-view, high resolution camera; Live Cell Chamber; and
the new Confocal Module, while our software products like Thermo Scientific™ HCS Studio™ Software enable
users to develop and make decisions about our assays. With the Thermo Scientific™ HCS101 Class and the
diverse portfolio of reagents and consumables, we enable scientists to increase their efficiency with their platform
while generating more knowledge about the cell.
More knowledge evolved into products
• More out-of-the-box reagents validated
for high content
• More flexibility in instrumentation to
address new assay needs
More knowledge about cellular information
• More measurements and data about cells
and their response
• More information than other cell-based assays
Cellular
Information
Validated
SOlutions
Contextual
Relevance
more
Knowledge
More knowledge in how to execute HCA
• More technical resources focused on
high content
• More experience in using, developing,
and executing on high content assays
Experienced
Execution
Scientific
Validity
More knowledge about cells in their context
• More information in the context of a
living cell
• More tools to characterize complex
biologies
Scientific validity through literature
• More peer-reviewed publications in the
most relevant and respected journals
• Automated solutions to increase throughput
For more information on Thermo Scientific™ High Content Products, and a full list of applications by application
area, go to thermoscientific.com/highcontent
Appl i cati o n No tes C- AN_ NT 1 1 1 2
Monitoring Neurite Morphology and
Synapse Formation in Primary Neurons
for Neurotoxicity Assessments and Drug
Screening
Suk J. Hong and Richik N. Ghosh
Thermo Fisher Scientific • Pittsburgh, PA USA
% Maximum
100
80
60
40
1
** **
10
100
1000
utamate [µM]
synaptic intensity
stsynaptic intensity
20
0.01
0.1
1
10
100
1000
Glutamate [µM]
Presynaptic vesicl
Postsynaptic spots
Synapse number
120
% Maximum
100
80
** *
60
40
20
0.1
1
10
0
0.001
100
0.01
inate [µM]
0.1
1
10
100
Kainate [µM]
Presynaptic vesicl
Postsynaptic spots
Synapse number
synaptic intensity
tsynaptic intensity
120
% Maximum
100
80
60
40
**
20
1
1
10
H2O2 [µM]
0
100 1000
0.001 0.01
0.1
1
10
100 1000
H2O2 [µM]
Presynaptic vesicl
Postsynaptic spots
Synapse number
synaptic intensity
tsynaptic intensity
120
% Maximum
100
80
60
**
1
10
40
0.01
synaptic intensity
tsynaptic intensity
**
20
100 1000
ZnCl2 [µM]
120
0.1
1
10
100
ZnCl2 [µM]
1000
Presynaptic vesicl
Postsynaptic spots
Synapse number
% Maximum
100
80
60
40
20
0.1
1
10
U0126 [µM]
100
0
0.001
0.01
0.1
1
10
U0126 [µM]
100
Introduction
B.
Presynaptic Marker
Neurons in central DAPI
and peripheral
nervous systems
function to transmit electric signals from one
location
• Rat Hippocampal
neurons 22 DIV
to the other to keep the brain and the body functioning
• Map2 (green)
properly. One of the critical structures in the
neuron to
• Synaptophysin (red)
maintain their proper functional network •is Imaged
synapse,
on ArrayScan
HCSthe
Reader
which is the junction between a nerve cell and
cell
that receives an impulse from the neuron. The molecular
MAP-2
network between these synapses controls not just
synaptic signal transmission and synaptic plasticity
but also regulates neuronal growth, differentiation and
death. The microstructure of synaptic junctions has
been extensively studied to understand the relationship
between synaptic activity and neuropathophysiology,
as well as the molecular mechanism involved in
synaptogenesis and the regulation of synapse.
Once synaptic function is disrupted by natural or
man-made neurotoxic substances, it could lead to
long-lasting and often irreversible neuronal damage.
Synaptic damage has often been recognized as the first
sign of neurodegeneration in many different pathological
Raw Im
Presyna
Neurona
Automated Data
Management
Decisions
Thermo Scientific HCSExplorer
(Thermo Scientific Store)
Analysis & Visualization
– vHCS Discovery ToolBox
Synaptogenesis Assay Target Candidates
Fluorescence
Channel:
Cellular
EntityTargeted:
Channel 1
Channel 2
Channel 3
Channel 4
Nucleus
Cell Body /
Neurite Mask
Postsynaptic
marker
Presynaptic
marker
PSD95, drebrin,
spinophilin/Neur
abin
synaptophysin,
synapsin1,
syntaxin,
synaptobrevin,
synaptotagmin
DyL549
DyL649
Neuronal
Neurite T
Candidates for
Cellular Target:
(best assay target
screened in bold)
DNA
Fluorescence
Dye & Color:
DAPI
MAP-2,
β3-tubulin,
Neurofilament
DyL488
Table 1: Potential synaptogenesis HCS assay targets can be
***
detected in four different colors.
0.400
15 DIV
0.350
21 DIV
0.300
0.250
0.200
125
% Maximum
120
(BioApplications)
100
75
50
25
0
0.150
06
2
0. 5
12
5
Presynaptic vesicl
Postsynaptic spots
Synapse number
conditions, including traumatic nerve injury, ischemic
stroke, and many neurodegenerative disorders such as
Motor Neuron Disease, Alzheimer’s, Parkinson’s and
Huntington’s diseases. Many synaptic proteins play an
important role in the progression of neurodegenerative
diseases. For example, Amyloid beta precursor protein
and Presenilin, alpha-synuclein, Huntingtin, Ataxin-1,
Frataxin and Prion protein are all involved in presynaptic or post-synaptic structure of the neuron and
play a role in synaptic damage and neurodegeneration.
To measure the synaptic changes that occur in
synaptogenesis or synaptic damage, we needed to
develop a reliable, accurate, and efficient method to
measure accurate synaptic loss, neurite changes and
neuronal death. Here we introduce a new way of
measuring synaptic function utilizing the power of
automated, quantitative, high-content cell-based imaging
Real Time Quantitative Image
Automated
Image Acquisition
and
analysis.
The Thermo
Scientific
Synaptogenesis
Automated
Plate Delivery
Analysis
ArrayScan VTI HCS Reader
HCS
Assay Reagents combined with the Thermo
Scientific ArrayScan High Content Screening (HCS)
Reader and Neuronal Profiling BioApplication
enables the quantitation of neuronal morphology
and synapses in vitro. On-the-fly automated image
analysis and quantitation accompanying the automated
image acquisition is done by the Neuronal Profiling
BioApplication, which is an automated image analysis
software module on the ArrayScan™ VTI HCS Reader.
Using this technology and assay method, we could
identify synaptic changes over time and measure synaptic
and neurite parameters in an automated manner.
0.
synaptic intensity
stsynaptic intensity
Synapse formation during nervous system development and degeneration
in the pathogenesis of human neurological diseases are highly regulated
processes. Subtle changes in the environment of the complex neuronal
network may cause either breakdown or creation of synaptic connections.
Drug discovery screening for neurological diseases and compound
neurotoxicity evaluation would benefit from robust, automated, quantitative
in vitro assays that monitor neuronal function. We hypothesized that
(1) toxic insults to the nervous system will cause neuronal synapses to
deteriorate in the early phase of neurotoxicity, eventually leading to neurite
degeneration and neuronal cell death if the damage is severe; and (2) an
in vitro functional assay for synapse formation and neuronal morphology
could be used to monitor and identify such neurotoxic events. We thus
developed an automated, functional, high-content screening imaging assay
to track and quantify the dynamic changes in neurite morphology and
synapses. This assay identifies primary neuronal cells by a neuron-specific
marker and detects synapses on the spines of neurites with pre- and postsynaptic markers. The multiplexed targets, including a nuclear marker, are
simultaneously detected with four fluorescent colors, and the fluorescent
A.
DAPI
images of the labeled neurons
and synapses
are acquiredWCS
by an automated
imaging instrument. The phenotypic features of neuronal morphology
• Mouse cortical
neurons 18DIV
and the synapse are automatically identified and quantified on-the-fly
by
• Cellomics Whole
specialized image-analysis software. Such features are potential
indicators
Cell Stain (red)
for neuronal development, differentiation and neurotoxicity, and• we
could
Synaptophysin
quantify changes in these features under different conditions and(green)
for different
Imaged on
drug treatments. By monitoring changes in these features, we •could
also
ArrayScan HCS
Presynaptic Marker
quantitatively evaluate compounds involved in developmental neurotoxicity.
Reader
In summary, this assay facilitates automation and streamlining of a
laborious process in drug discovery screening and compound neurotoxicity
** *
assessments;
it enables quantitative comparisons between compounds
in neuronal morphology and function, particularly in neurite and synapse
associated events.
Spot Number per Neurite Length
Abstract
0.100
0.050
0.000
Presynaptic
Postsynaptic
Automated Image Acquisition
Automated Plate Delivery
ArrayScan VTI HCS Reader
Automated Image Acquisition
ortical
18DIV
s Whole
n (red)
physin
Real Time Quantitative Image
Analysis (BioApplications)
Real Time Quantitative Image
Analysis (BioApplications)
on
Raw Image
an HCS
use
cortical
rons 18DIV
omics Whole
Stain (red)
Automated Data
Decisions
Management
aptophysin
Analysis & Visualization
–
en)
ged on Fluorescence
Channel 1
Channel 2
Channel 3
Channel 4
Channel:
ayScan
HCS
Pre-Synaptic
Marker, Synaptophysin, Whole
campal
2der
DIV
Postsynaptic
Presynaptic
Cellular
Cell
Stain andNucleus
MAP-2 Cell Body /
en)
marker
Raw Image
Presynaptic Marker Syna
Neuronal Marker MAP-2
Real Time Quantitative Image
Analysis (BioApplications)
ArrayScan VTI HCS Reader
Neuronal Marker, MAP-2 (white)
Neurite Trace from Cell Body (Blue)
Automated
Data
WCS
synaptophysin,
Management
PSD95,
drebrin, synapsin1,
DAPI
Candidates for
Cellular Target:
DNA
& Visualization
(bestAnalysis
assay target
screened
in bold)
Discovery ToolBox
– vHCS
Figure 2: Mouse cortical neurons (14 DIV) were stained for
synaptophysin (red) and MAP-2 (green) (left panel), and
analyzed (right panel) with the ArrayScan VTI HCS Reader and
the Neuronal Profiling
v3.5 BioApplication. Automated Image Acquisition
Automated Plate Delivery
Postsynaptic Marker, PSD-95 (magenta)
Co-localization (green) of PSD-95 and Synapt
MAP-2,
(Thermo
Scientific Store)
• Mouse cortical
β3-tubulin,
spinophilin/Neur
syntaxin,
Neurofilament
abin
synaptobrevin,
neurons 18DIV
synaptotagmin
•
Automated, Simultaneous Measurement of
PresynapticAutomated
Vesicles,
Postsynaptic
Structure
Automated
Image Acquisition
Plate Delivery
Cellomics Whole
ArrayScan VTI HCS Reader
and
Neurites
Cell Stain (red)
Real Time Quantitative Image
Analysis (BioApplications)
Aβ1-42 Toxicity on Primary Hippocampal Neuron (50DIV)
DyL549WCS
DyL649
DAPI DyL488
Automated Data
Real Time Quantitative Image• Synaptophysin
Decisions
Automated Image Acquisition
Management
(BioApplications)
Channel Thermo
2
Channel
3AnalysisChannel
4 • (green)
Scientific
HCSExplorer
Mouse cortical
Presynaptic vesicles
Presynaptic intensity
Neuron number
Neurite Intensity
Branch Point
(Thermo
Scientific Store)
0.400
Postsynaptic spots
• *** Imaged
on
Neurite Count
Postsynaptic intensity
Neurite Total Length
Neurite
Width
neurons 18DIV
Analysis & Visualization
Synapse number
15 DIV
0.350
ArrayScan HCS
21 DIV
– vHCS Discovery ToolBox
• Cellomics Whole
0.300
Presynaptic Marker
**
Reader
**
* * *
Cell Body /
Postsynaptic
Presynaptic
0.250
***
Cell Stain (red)
**
**
Neurite Mask
marker0.200
marker
• Synaptophysin
0.050
0.000
60
Channel 2
80
60
40
Channel 4
8
16
32
5
2
4
0.
0.
Aβ 1-42 µM]
06
2
0. 5
12
5
0.
25
8
32
16
1
2
4
25
0.
5
5
25
12
0
0.
06
32
4
8
0.
0.
1-42
Channel 3
16
5
1
2
0.
06
2
0. 5
12
5
0.
25
8
16
32
5
Aβ 1-42
Postsynaptic Marker, PSD-95 (magenta)
of
PSD-95 and Synapt
µM] Co-localization (green)
µM]
Aβ
4
1
0.
0.
0
2
06
2
0. 5
12
5
0.
25
4
8
16
32
5
2
1
0.
1-42
30
20
0
Neuronal Marker, MAP-2 (white)
Neurite Trace Aβ
from Cell
µM]Body (Blue)
100
60
1
25
80
Maximum
50
120
90
%
100
75
0.
25
% Maximum
% Maximum
50
(green)
• Imaged on
synaptophysin,
ArrayScan HCS
Presynaptic
Postsynaptic
synapsin1,
Reader
Channel 1
120
100
75
0
Fluorescence
Channel:
Presynaptic
Markerdrebrin,
PSD95,
0.100
100
06
2
0. 5
12
5
0.
25
0.150
120
125
% Maximum
% Maximum
125
0.
Spot Number per Neurite Length
MAP-2,
DAPI
]
Dye & Color:
]
A.Fluorescence
]
ucleus
A.
A.
marker
Analyzed Image
Branch point (white)
Localized Synaptophysin (purple)
Neuronal trace (blue)
]
nnel 1
Raw Image
vHCS Discovery ToolBox
Neurite Mask
Branch point (white)
Localized Synaptophysin (purple)
Neuronal trace (blue)
Presynaptic Marker Synaptophysin (red)
Neuronal Marker MAP-2 (green)
(Thermo Scientific Store)
EntityTargeted:
Analyzed Image
Presynaptic Marker Synaptophysin (red)
Neuronal Marker MAP-2 (green)
Aβ 1-42 µM]
]
ysin (red)
ArrayScan
er
Thermo Scientific HCSExplorer
Real Time Quantitative Image
Analysis (BioApplications)
Automated Image Acquisition
Automated Plate Delivery
ArrayScan VTI HCS Reader
Appl i cati o n No tes C- AN_ NT 1 1 1 2
Automated Measurement of Presynaptic
Vesicles and Neurites Using The Thermo
Scientific Neuronal Profiling V3.5
The Thermo Scientific HCS Platform Seamless
Integration of all the Steps in Cellular Analysis
Automated Data
0.100
0.050
0.000
0.300
0.000
0.250
8
2
4
1
25
0.
5
5
25
12
06
0.
0.
0.
***
100
DyL649
75
50
25
4
2
1
***
5
0
0.
21 DIV
125
06
2
0. 5
12
5
0.
25
15 DIV
synaptophysin,
Aβ 1-42 µM]
DyL649
synapsin1,
syntaxin,
synaptobrevin,
Neuron number
synaptotagmin
Neurite Count
0.
0.150
0.150
0.400
0.100
0.350
0.050
% Maximum
32
8
16
4
1
2
5
0.
2
0. 5
12
5
0.
25
06
8
4
16
32
0.
DyL549
0.250
Postsynaptic Marker, PSD-95
0.200 (magenta)
Co-localization (green) of PSD-95 and Synapt
0.200
Presynaptic
Aβ 1-42 µM]
Postsynaptic
0.200
0.150
0.100
0.050
0.000
Figure 4: Mouse cortical neurons were cultured for 15 DIV orPresynaptic
21 DIV and stained for synaptophysin, PSD- 95 and MAP-2,
imaged and analyzed. Only postsynaptic spots stained with
numberincreases in 21 Neurite
Intensity compared toBranch
Point
PSD-95Neuron
antibody
DIV neurons
15 DIV
Neurite
Count
Neurite
Total Length spots show
Neurite
neurons
(Student’s
t-test, p<0.001).
Presynaptic
no Width
significant change.
125
% Maximum
er Neurite Length
0.400
0.200
0.300
0
Postsynaptic
Aβ1-42 Toxicity
on Primary Hippocampal Neuron
Figure 1: A. Synaptophysin is a good presynaptic marker. DAPI and
Whole Cell Stain is used to detect the nuclei and the fine structure
of neurites, respectively (Mouse cortical neuron, 18DIV).
0.250
21 DIV
DyL488
Presynaptic
DyL649
***
15 DIV
B. Synaptophysin
and MAP-2 staining
for presynaptic
vesicle and
0.350
21 DIV neuron, 22DIV).
neurite detection (Rat hippocampal
2
DAPI
15 DIV
0.350
90
60
syntaxin,
Presynaptic
synaptobrevin,
30
marker
synaptotagmin
100
75
50
25
***
75
50
25
Presynaptic intens
Postsynaptic inten
120
125
100
Postsynaptic
100
**
**
80
60
120
% Maximum
• Synaptophysin (red)
• Imaged on the ArrayScan VTI HCS Reader
0.300
5
0.
0.
Fluorescence
Dye & Color:
% Maximum
synaptophysin,
MAP-2,
PSD95, drebrin, synapsin1,
β3-tubulin,
spinophilin/Neur syntaxin,
abin
synaptobrevin,
•Neurofilament
Rat Hippocampal
Neurons 22 DIV
synaptotagmin
• Map-2 (green)
DyL549
1
06
2
0. 5
12
5
0.
25
16
32
4
8
5
2
1
0.
5
25
12
0.
0.
0.300
21 DIV
Neuronal
0.250Marker, MAP-2 (white)
Neurite Trace from Cell Body (Blue)
DyL488
% Maximum
% Maximum
% Maximum
25
06
0.
Presynaptic
marker
***
15 DIV
% Maximum
Postsynaptic
marker
0.350
Spot Number per Neurite
Spot
Length
Number per Neurite Length
Cell Body /
Neurite Mask
Channel
3
MAP-2
Presynaptic
75
% Maximum
0.000
Channel
2
75
Spot Number per Neurite Length
0.050
spots (green spot).
Co-localized
spots (green)**represent
the
Rat Hippocampal
Cellular
Target:
80
50
50
DNA
β3-tubulin,
spinophilin/Neur
***synapses.
neurons 22 DIV
location
of potential
(best
assay
target
Cell Body /
Postsynaptic
Cellular
Neurofilament
abin
(Thermo Scientific Store)
25
25
Nucleus
screened in bold)
• Map2 (green)
**Neurite60 Mask
marker
EntityTargeted:
0
0
• Synaptophysin (red)
DAPI
DyL488
DyL549
DyL649
• Imaged on ArrayScan
Fluorescence
Aβ 1-42 µM]
AβDAPI
µM]
µM]
Aβ 1-42DyL549
HCS Reader
Candidates for
1-42
DyL488
PunctatedDye
PSD-95
Stain Increases
by
MAP-2,
PSD95, drebrin,
& Color:
Cellular
Target:
DNA
β3-tubulin,
spinophilin/Neur
Maturation
of
Neurons
(best
assay
target
Channel 4
Neurofilament
abin
screened in bold)
Postsynaptic
0.400
0.400
Automated Data •
Management
]
0.100
Dye & Color:
100
]
DAPI
MAP-2
0.200
Analysis
& Visualization
Fluorescence
vHCS Discovery ToolBox
– 0.150
120
100
]
DNA
0.250
120
125
100
]
ucleus
125
]
annel 1
Spot Number per Neurite Length
Decisions
DNA
β3-tubulin,
spinophilin/Neur syntaxin,
Management
Hippocampal
Thermo Scientific HCSExplorer
(Thermo Scientific Store)
Neurofilament
abin
synaptobrevin,
Analysis & Visualization
ons 22 DIV
– vHCS Discovery ToolBox
Postsynaptic
Presynaptic
Cellular
Cell Body /
synaptotagmin
Nucleus
(green) • Mouse corticalEntityTargeted:
Raw Image
marker Analyzed Image
Mask
marker
Neurite
neurons 18 DIV
Presynaptic Marker Synaptophysin (red)
Branch point (white)
B.
Neuronal Marker, MAP-2 (w
DAPI Cell Stain
Presynaptic
ptophysin•(red)
Thermo Scientific Whole
(red)Marker
Neuronal Marker MAP-2
(green)
Localized Synaptophysin (purple)
Fluorescence
Neurite
Trace
Aβ
Toxicity
on Primary
Hippocampal
Neuron
Automated
Data
Channel
1trace (blue)
Channel
2
Channel
3
Channel
4from Cell Bod
Decisions
1-42Neuronal
DAPI
DyL488
DyL549
DyL649
Management
Channel:
• Synaptophysin (green)
ed on ArrayScan
Thermo
Scientific HCSExplorer
(Thermo Scientific Store)
synaptophysin,
• Rat Hippocampal
& Visualization
Figure 3: Rat hippocampal neurons (21 DIV) were Analysis
stained
for
VTI
Reader • Imaged on the ArrayScan
Candidates
forHCS Reader
neurons 22 DIV
– vHCS Discovery ToolBox
Body /
Postsynaptic
Presynaptic
Cellular
MAP-2,
PSD95,
drebrin,
synapsin1,
synaptophysin,
PSD-95
and MAP-2,
imaged
andCell
analyzed.
Nucleus
Cellular Target:
• Map2 (green)
Presynaptic intens
marker
marker
EntityTargeted:
Neuron number
Neurite Intensity Neurite MaskBranch Point
DNA
β3-tubulin,
spinophilin/Neur
syntaxin,
0.400
Left
panel:
detection with
MAP-2
staining.
B.
• Synaptophysin
(red)
NeuriteNeurite
Count
Postsynaptic inten
Neurite
Total
Length
Neurite Width
(bestDAPI
assay
target ***
B.
15 DIV Presynaptic Marker
Fluorescence
Neurofilament
abin marker
synaptobrevin,
Right panel: Postsynaptic
spot detection
with PSD-95
0.350
• Imaged on
ArrayScan
Channel
1
Channel
2
Channel
3
Channel
4
screened 21
inDIVbold)
synaptophysin,
spots)for
and co-localization
with synaptophysin
Channel:
HCS Reader staining (magenta
Candidates
synaptotagmin
0.300
MAP-2,
PSD95, drebrin, synapsin1,
90
60
30
Channel 4
1
10
C
0.001
0.01
25
10
0.001 0.01 0.1
1
10
50
**
0
H2O2 [µM]
0.001 0.01
50
25
0.1
1
10
100
ZnCl2 [µM]
***
100
80
60
1000
*
0.01
1
10
100
% Maximum
50
25
*
75
50
25
0.001 0.01
0.1
1
10
U0126 [µM]
0.01
100
0
0.1
1
10
100
ZnCl2 [µM]
0.01
0.1
1
10
100
U0126 [µM]
0.001 0.01
0.1
1
10
10
100
% Maximum
0
100 1000
0.001 0.01
1
10
120
60
**
30
0.1
1
10
40
Postsynaptic intensity
**
20
100 1000
0.01
ZnCl2 [µM]
120
0.1
1
10
100
ZnCl2 [µM]
1000
Presynaptic vesicl
Postsynaptic spots
Synapse number
100
90
80
60
60
Maximum
%
20
30
20
0.001 0.01
0.1
1
10
U0126 [µM]
100
0
0.001
0.01
0.1
1
10
100
U0126 [µM]
Map2 (green)
Synaptophysin (red)
Imaged on ArrayScan
HCS Reader
MAP-2
Summary
8
Aβ 1-42 µM]
Cellular
EntityTargeted:
Nucleus
Candidates for
Cellular Target:
(best assay target
screened in bold)
DNA
Fluorescence
Dye & Color:
DAPI
Multiparameter Synaptogenesis Assay simultaneously
identifies and quantifies neurites, pre- and post-synaptic
structures and synapse in an automated manner.
• Neurotoxicity from neurotoxic substances is accurately
detected.
• Substances only affecting synapse can be detected.
• Assay works for acute or chronic neurodegenerative
disease cell models.
thermoscientific.com/highcontent
© 2013 Thermo Fisher Scientific Inc. All rights reserved. Alexa Fluor is a trademark of Molecular Probes, Inc. Packard is a trademark of Perkin Elmer Inc. X-Light is a
trademark of Crisel Electrooptical Systems & Technology s.r.l. All other trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries. Specifications,
terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details.
USA +1 800 432 4091 [email protected]
Asia +81 3 5826 1659 [email protected]
Europe +32 (0)53 85 71 84 [email protected]
32
2
4
16
0
1
32
40
0.1 5
25
0.2
5
0.5
16
* * *
60
62
1
2
8
4
80
0.0
% Maximum
5
0.1
25
0.2
5
0.5
32
62
1
4
2
100
40
Figure 5: Mouse, rat cortical or hippocampal primary neurons
were cultured for 21 DIV, and the dose dependent responses
of drugs towards various properties of these neurons were
investigated. (A) Glutamate with 10 mM glycine in HBSS was
treated for 30 min, washed and replaced with culture media.
After 24 hr incubation, neurons were fixed, stained and
analyzed. (B) Kainate, (C) H2O2, (D) Zinc, (E) U0126 were treated
for 24 hrs in culture media. (Student’s t-test, *p<0.05, **p<0.01,
***p<0.001).
C-AN_NT1112
30
0.0
1
4
8
16
2
Aβ 1-42
•
•
•
H2O2 [µM]
80
60
Presynaptic vesicles
Postsynaptic spots
Synapse number
120
Automated Data
Management
Decisions
100 1000
100
60
60
Presynaptic vesicl
Postsynaptic spots
Synapse number
Postsynaptic intensity
90
0
0.1
80
90
Thermo Scientific HCSExplorer
**
20
10
120
Figure 6: Rat hippocampal primary neurons were cultured for
50 DIV. Dose dependent responses of Aβ1-42 aggregates were
investigated. 500 mM Aβ1-42 was incubated at 37 °C in media
for 3 days to induce oligomerization. Neurons were incubated
withB.the Aβ1-42 oligomers for 48 hrs, and then fixed, stained, and
DAPI
Presynaptic Marker
Fluorescence
analyzed. Aβ1-42 toxicity
leads
to synapse loss. (Student’s
t-test,
Channel 1
Channel:
• Rat Hippocampal
*p<0.05, **p<0.01, ***p<0.001).
neurons 22 DIV
60
1
Presynaptic intensity
Mouse cortical
Postsynaptic intensity
neurons 18DIV
• Cellomics Whole
Cell Stain (red)
**
• Synaptophysin
**
(green)
• Imaged on
µM] ArrayScan HCS
Aβ 1-42 µM]
Reader
0
Aβ 1-42 µM]
Presynaptic Marker
Presynaptic vesicl
Postsynaptic spots
Synapse number
120
% Maximum
**
25
0.0
4
8
32
2
16
1
62
0.1 5
25
0.2
5
0.5
% Maximum
U0126 [µM]
1
40
0.01
100
0.1
**
50
0
Aβ 1-42 µM]
Branch Point•
Neurite Width
62
0.1 5
25
0.2
5
0.5
25
0.0
***
50
120
100
75
8
16
32
% Maximum
100
75
0.0
0.01
Kainate [µM]
Presynaptic intensity
80
100
Neurite Intensity
Neurite Total Length
80
0.001 0.01 0.1
120
*
125
100
1000
60
0.001
0
0.001
100
30
H2O2 [µM]
Neurite Width
100
10
Postsynaptic intensity
Branch Point
120
1
60
60
1000
** *
40
120
**
** *
100
20
0.1
Presynaptic intensity
Neurite Width
10
60
30
0
1
80
60
90
100 1000
80
1000
Neurite Total Length
100
75
10
100
Neurite Intensity
Neurite Count
100
0
0.1
1
0.1
Glutamate [µM]
Presynaptic vesicl
Postsynaptic spots
Synapse number
120
Branch Point
120
ZnCl2 [µM]
Neuron number
125
0.001 0.01 0.1
1000
0.01
Presynaptic intensity
H2O2 [µM]
40
0.01
E
100
Neurite Total Length
120
% Maximum
*
75
10
Neurite Intensity
*
100
1
H2O2 [µM]
Neuron number
Neurite Count
0.1
20
1000
100
120
** **
60
100
Kainate [µM]
*
80
** **
10
Glutamate [µM]
0
0.001 0.01
Neurite Width
100
1
Presynaptic intensity
Postsynaptic intensity
Branch Point
120
0.1
90
100
Postsynaptic
40
0.01
Kainate [µM]
75
100 1000
10
80
60
30
120
* *
1
100
60
100 1000
90
0.1
120
Presynaptic
90
Glutamate [µM]
80
0.001 0.01
100
** **
100
25
125
% Maximum
1
Neurite Total Length
% Maximum
% Maximum
H2O2
50
D
Zinc
**
75
% Maximum
0.1
Neurite Intensity
100
10
Branch Point
Neurite Width
120
Kainate [µM]
Neurite Count
1
100
* *
60
Neuron number
125
0.1
110
80
100
120
0.000
Presynaptic vesicl
Postsynaptic spots
Synapse number
Presynaptic intensity
Postsynaptic intensity
0.050
WCS
DAPI
Neuron number
Neurite Count
Real Time Quantitative Image
Analysis (BioApplications)
Automated Image Acquisition
(Thermo Scientific Store)
Analysis & Visualization
– vHCS Discovery ToolBox
Channel 2
Channel 3
Channel 4
Cell Body /
Neurite Mask
Postsynaptic
marker
Presynaptic
marker
PSD95, drebrin,
spinophilin/Neur
abin
synaptophysin,
synapsin1,
syntaxin,
synaptobrevin,
synaptotagmin
DyL549
DyL649
MAP-2,
β3-tubulin,
Neurofilament
DyL488
Spot Number per Neurite Length
0.1
0.100
Postsynaptic Marker, PSD-95 (magenta)
Co-localization (green) of PSD-95 and Synapt
Automated Plate Delivery
Aβ1-42 Toxicity on Primary Hippocampal Neuron (50DIV)
ArrayScan VTI HCS Reader
0
0.150
% Maximum
0
0.001 0.01
0.200
% Maximum
*
25
0.01
125
0.250
% Maximum
% Maximum
50
80
1000
Neurite Intensity
Neurite Total Length
Kainate [µM]
U0126
100
100
75
0
10
Glutamate [µM]
120
100
1
Neuronal Marker, MAP-2 (white)
Neurite Trace from Cell Body (Blue)
***
15 DIV
% Maximum
Glutamate [µM]
0.1
Neurite and Synapse Changes as Neurotoxicity
Response Against Aβ1-42 Aggregates
A.
21 DIV
0.300
% Maximum
0.01
DyL649
0.350
60
60
Neuron number
Neurite Count
125
80
100 1000
% Maximum
10
% Maximum
1
% Maximum
0.1
DyL549
0.400
% Maximum
100
% Maximum
*
0.01
PSD95, drebrin,
spinophilin/Neur
abin
synaptophysin,
synapsin1,
syntaxin,
synaptobrevin,
synaptotagmin
DyL488
Branch Point
Neurite Width
120
Presynaptic
marker
% Maximum
Neurite Intensity
Neurite Total Length
Postsynaptic
marker
% Maximum
DAPI
MAP-2,
β3-tubulin,
Neurofilament
Spot Number per Neurite Length
Fluorescence
Dye & Color:
% Maximum
% Maximum
50
25
% Maximum
DNA
100
75
Cell Body /
Neurite Mask
Nucleus
Candidates for
Cellular Target:
(best assay target
screened in bold)
120
100
B
Kainate
Neuron number
Neurite Count
125
% Maximum
Glutamate
A
Cellular
EntityTargeted:
]
•
•
•
Appl i cati o n No tes C- AN_ NT 1 1 1 2
Neurite and Synapse Changes as Neurotoxicity
Response Against Drug Treatments
% Maximum
Channel 3
62
0.1 5
25
0.2
5
0.5
Channel 2
]
Channel 1
]
Fluorescence
Channel:
Rat Hippocampal
neurons 22 DIV
Map2 (green)
Synaptophysin (red)
Imaged on ArrayScan
HCS Reader
]
•
]
Presynaptic Marker
0.400
15 DIV
0.350
***
21 DIV
0.300
0.250
0.200
0.150
0.100
0.050
0.000
Presynaptic
Postsynaptic
Neuroscience Publications List
Bajenaru, M.L. et al. β1 Integrin-Focal Adhesion Kinase (Fak) Signaling Modulates Retinal Ganglion Cells Survival.
Invest. Ophthalmol. Vis. Sci. 53, 3482- (2012).
Bao, R. et al. Targeting Heat Shock Protein 90 with Cudc-305 Overcomes Erlotinib Resistance in Non-Small Cell
Lung Cancer. Mol. Cancer Ther. 8, 3296-3306 (2009).
Bauer, P.O. et al. Inhibition of Rho Kinases Enhances the Degradation of Mutant Huntingtin. J. Biol. Chem. 284,
13153-13164 (2009).
Blackmore, M.G. et al. High Content Screening of Cortical Neurons Identifies Novel Regulators of Axon Growth.
Mol. Cell. Neurosci. 44, 43-54 (2010).
Blackmore, M.G. et al. Krüppel-Like Factor 7 Engineered for Transcriptional Activation Promotes Axon
Regeneration in the Adult Corticospinal Tract. Proc. Natl. Acad. Sci. U. S. A. 109, 7517-7522 (2012).
Borikova, A.L. et al. Rho Kinase Inhibition Rescues the Endothelial Cell Cerebral Cavernous Malformation
Phenotype. J. Biol. Chem. 285, 11760-11764 (2010).
Breier, J.M., Radio, N.M., Mundy, W.R. & Shafer, T.J. Development of a High-Throughput Screening Assay for
Chemical Effects on Proliferation and Viability of Immortalized Human Neural Progenitor Cells. Toxicol. Sci. 105,
119-133 (2008).
Brickelmaier, M. et al. Identification and Characterization of Mefloquine Efficacy against Jc Virus in Vitro.
Antimicrob. Agents Chemother. 53, 1840-1849 (2009).
Buchser, W.J., Pardinas, J.R., Shi, Y., Bixby, J.L. & Lemmon, V.P. 96-Well Electroporation Method for Transfection
of Mammalian Central Neurons. BioTechniques 41, 619-624 (2006).
Buchser, W.J., Slepak, T.I., Gutierrez-Arenas, O., Bixby, J.L. & Lemmon, V.P. Kinase/Phosphatase Overexpression
Reveals Pathways Regulating Hippocampal Neuron Morphology. Mol. Syst. Biol. 6, 391 (2010).
Chantong, B., Kratschmar, D.V., Nashev, L.G., Balazs, Z. & Odermatt, A. Mineralocorticoid and Glucocorticoid
Receptors Differentially Regulate NF-κB Activity and Pro-Inflammatory Cytokine Production in Murine BV-2
Microglial Cells. J. Neuroinflammation 9, 260 (2012).
Clement, C.M., Dandepally, S.R., Williams, A.L. & Ibeanu, G.C. A Synthetic Analog of Verbenachalcone
Potentiates Ngf-Induced Neurite Outgrowth and Enhances Cell Survival in Neuronal Cell Models. Neurosci. Lett.
459, 157-161 (2009).
Culbert, A.A. et al. Mapk-Activated Protein Kinase 2 Deficiency in Microglia Inhibits Pro-Inflammatory Mediator
Release and Resultant Neurotoxicity: Relevance to Neuroinflammation in a Transgenic Mouse Model of Alzheimer
Disease. J. Biol. Chem. 281, 23658-23667 (2006).
Dahl, J.P. et al. Characterization of the Wave1 Knock-out Mouse: Implications for Cns Development. J. Neurosci.
23, 3343-3352 (2003).
del Valle, K., Theus, M.H., Bethea, J.R., Liebl, D.J. & Ricard, J. Neural Progenitors Proliferation Is Inhibited by
Ephb3 in the Developing Subventricular Zone. Int. J. Dev. Neurosci. 29, 9-14 (2011).
Denis, J.A. et al. Mtor-Dependent Proliferation Defect in Human ES-Derived Neural Stem Cells Affected by
Myotonic Dystrophy Type 1. J. Cell Sci. 126, 1763-1772 (2013).
Doi, H. et al. Rna-Binding Protein Tls Is a Major Nuclear Aggregate-Interacting Protein in Huntingtin Exon 1 with
Expanded Polyglutamine-Expressing Cells. J. Biol. Chem. 283, 6489-6500 (2008).
Doumanis, J., Wada, K., Kino, Y., Moore, A.W. & Nukina, N. Rnai Screening in Drosophila Cells Identifies New
Modifiers of Mutant Huntingtin Aggregation. PLoS One 4 (2009).
Draghetti, C. et al. Functional Whole-Genome Analysis Identifies Polo-Like Kinase 2 and Poliovirus Receptor
as Essential for Neuronal Differentiation Upstream of the Negative Regulator αB-Crystallin. J. Biol. Chem. 284,
32053-32065 (2009).
Drew, K.L., McGee, R.C., Wells, M.S. & Kelleher-Andersson, J.A. Growth and Differentiation of Adult Hippocampal
Arctic Ground Squirrel Neural Stem Cells. J. Vis. Exp. (2011).
Du, W., Hozumi, N., Sakamoto, M., Hata, J. & Yamada, T. Reconstitution of Schwannian Stroma in
Neuroblastomas Using Human Bone Marrow Stromal Cells. Am. J. Pathol. 173, 1153-1164 (2008).
Ehrnhoefer, D.E. et al. A Quantitative Method for the Specific Assessment of Caspase-6 Activity in Cell Culture.
PLoS One 6 (2011).
Emery, A.C. & Eiden, L.E. Signaling through the Neuropeptide Gpcr Pac1 Induces Neuritogenesis Via a Single
Linear Camp- and Erk-Dependent Pathway Using a Novel Camp Sensor. FASEB J. 26, 3199-3211 (2012).
Emery, A.C., Eiden, M.V. & Eiden, L.E. A New Site and Mechanism of Action for the Widely Used Adenylate
Cyclase Inhibitor SQ22,536. Mol. Pharmacol. 83, 95-105 (2013).
Eva, R., Bouyoucef-Cherchalli, D., Patel, K., Cullen, P.J. & Banting, G. Ip3 3-Kinase Opposes Ngf Driven Neurite
Outgrowth. PLoS One 7 (2012).
Falsig, J., Porzgen, P., Lotharius, J. & Leist, M. Specific Modulation of Astrocyte Inflammation by Inhibition of
Mixed Lineage Kinases with Cep-1347. J. Immunol. 173, 2762-2770 (2004).
Fennell, M., Chan, H. & Wood, A. Multiparameter Measurement of Caspase 3 Activation and Apoptotic Cell Death
in Nt2 Neuronal Precursor Cells Using High-Content Analysis. J. Biomol. Screen. 11, 296-302 (2006).
Foley, N.H. et al. Micrornas 10a and 10b Are Potent Inducers of Neuroblastoma Cell Differentiation through
Targeting of Nuclear Receptor Corepressor 2. Cell Death Differ. 18, 1089-1098 (2011).
Fowler, A. et al. A Nonradioactive High-Throughput/High-Content Assay for Measurement of the Human Serotonin
Reuptake Transporter Function in Vitro. J. Biomol. Screen. 11, 1027-1034 (2006).
Furukawa, Y., Kaneko, K., Yamanaka, K., O’Halloran, T.V. & Nukina, N. Complete Loss of Post-Translational
Modifications Triggers Fibrillar Aggregation of Sod1 in the Familial Form of Amyotrophic Lateral Sclerosis. J. Biol.
Chem. 283, 24167-24176 (2008).
Gao, Y. et al. Inhibition of Y-Box Binding Protein-1 Slows the Growth of Glioblastoma Multiforme and Sensitizes to
Temozolomide Independent O6-Methylguanine-DNA Methyltransferase. Mol. Cancer Ther. 8, 3276-3284 (2009).
Gaublomme, D., Buyens, T. & Moons, L. Automated Analysis of Neurite Outgrowth in Mouse Retinal Explants. J.
Biomol. Screen., 1087057112471989 (2012).
Gaughwin, P., Ciesla, M., Yang, H., Lim, B. & Brundin, P. Stage-Specific Modulation of Cortical Neuronal
Development by Mmu-Mir-134. Cereb. Cortex 21, 1857-1869 (2011).
ge, Y. et al. Promoting Retinal Ganglion Cell Axonal Regeneration by Inhibition of Transforming Growth Factor Beta
(Tgf{Beta}) Signaling. Invest. Ophthalmol. Vis. Sci. 52, 4617- (2011).
Gies, E. et al. Niclosamide Prevents the Formation of Large Ubiquitin-Containing Aggregates Caused by
Proteasome Inhibition. PLoS One 5 (2010).
Guan, J.S. et al. Hdac2 Negatively Regulates Memory Formation and Synaptic Plasticity. Nature 459, 55-60
(2009).
Hahn, A.T., Jones, J.T. & Meyer, T. Quantitative Analysis of Cell Cycle Phase Durations and PC12 Differentiation
Using Fluorescent Biosensors. Cell Cycle 8, 1044-1052 (2009).
Hansen, C. et al. A-Synuclein Propagates from Mouse Brain to Grafted Dopaminergic Neurons and Seeds
Aggregation in Cultured Human Cells. J. Clin. Invest. 121, 715-725 (2011).
Harrill, J.A. et al. Transcriptional Response of Rat Frontal Cortex Following Acute in Vivo Exposure to the
Pyrethroid Insecticides Permethrin and Deltamethrin. BMC Genomics 9, 546 (2008).
Henn, A., Kirner, S. & Leist, M. TLr2 Hypersensitivity of Astrocytes as Functional Consequence of Previous
Inflammatory Episodes. J. Immunol. 186, 3237-3247 (2011).
Hu, Y. et al. Netrin-4 Promotes Glioblastoma Cell Proliferation through Integrin B4 Signaling12. Neoplasia 14, 219227 (2012).
Hutson, T.H., Buchser, W.J., Bixby, J.L., Lemmon, V.P. & Moon, L.D.F. Optimization of a 96-Well Electroporation
Assay for Postnatal Rat Cns Neurons Suitable for Cost–Effective Medium-Throughput Screening of Genes That
Promote Neurite Outgrowth. Front. Mol. Neurosci. 4 (2011).
Ilyin, S.E. et al. Integrated Expressional Analysis: Application to the Drug Discovery Process. Methods 37, 280-288
(2005).
Jain, S., van Kesteren, R.E. & Heutink, P. High Content Screening in Neurodegenerative Diseases. J. Vis. Exp.
(2012).
Jepson, S. et al. Lingo-1, a Transmembrane Signaling Protein, Inhibits Oligodendrocyte Differentiation and
Myelination through Intercellular Self-Interactions. J. Biol. Chem. 287, 22184-22195 (2012).
Jossin, Y. & Cooper, J.A. Reelin, Rap1 and N-Cadherin Orient the Migration of Multipolar Neurons in the
Developing Neocortex. Nat. Neurosci. 14, 697-703 (2011).
Kaltenbach, L.S. et al. Composite Primary Neuronal High-Content Screening Assay for Huntington’s Disease
Incorporating Non-Cell-Autonomous Interactions. J. Biomol. Screen. 15, 806-819 (2010).
Kerrison, J.B., Duh, E.J., Yu, Y., Otteson, D.C. & Zack, D.J. A System for Inducible Gene Expression in Retinal
Ganglion Cells. Invest. Ophthalmol. Vis. Sci. 46, 2932-2939 (2005).
Kino, Y. et al. Intracellular Localization and Splicing Regulation of Fus/Tls Are Variably Affected by Amyotrophic
Lateral Sclerosis-Linked Mutations. Nucleic Acids Res. 39, 2781-2798 (2011).
Kuai, L. et al. Aak1 Identified as an Inhibitor of Neuregulin-1/Erbb4-Dependent Neurotrophic Factor Signaling
Using Integrative Chemical Genomics and Proteomics. Chem. Biol. 18, 891-906 (2011).
Kuai, L. et al. Chemical Genetics Identifies Small-Molecule Modulators of Neuritogenesis Involving Neuregulin-1/
Erbb4 Signaling. ACS Chem. Neurosci. 1, 325-342 (2010).
Kunzevitzky, N.J. et al. Foxn4 Is Required for Retinal Ganglion Cell Distal Axon Patterning. Mol. Cell. Neurosci.
46, 731-741 (2011).
Larsson, D.E., Hassan, S.B., Oberg, K. & Granberg, D. The Cytotoxic Effect of Emetine and Cgp-74514A Studied
with the Hollow Fiber Model and Arrayscan Assay in Neuroendocrine Tumors in Vitro. Anticancer Agents Med.
Chem. 12, 783-790 (2012).
Larsson, D.E., Wickstrom, M., Hassan, S., Oberg, K. & Granberg, D. The Cytotoxic Agents Nsc-95397, Brefeldin
a, Bortezomib and Sanguinarine Induce Apoptosis in Neuroendocrine Tumors in Vitro. Anticancer Res. 30, 149156 (2010).
Le, M.T. et al. Microrna-125b Is a Novel Negative Regulator of p53. Genes Dev. 23, 862-876 (2009).
Le, M.T.N. et al. Microrna-125b Promotes Neuronal Differentiation in Human Cells by Repressing Multiple Targets†.
Mol. Cell. Biol. 29, 5290-5305 (2009).
Lerch, J.K. et al. Isoform Diversity and Regulation in Peripheral and Central Neurons Revealed through Rna-Seq.
PLoS One 7 (2012).
Leyva, M.J. et al. Identification and Evaluation of Novel Small Molecule Pan-Caspase Inhibitors in Huntington’s
Disease Models. Chem. Biol. 17, 1189-1200 (2010).
Lie, M., Grover, M. & Whitlon, D.S. Accelerated Neurite Growth from Spiral Ganglion Neurons Exposed to the Rho
Kinase Inhibitor H-1152. Neuroscience 169, 855-862 (2010).
Lipinski, M.M. et al. Genome-Wide Analysis Reveals Mechanisms Modulating Autophagy in Normal Brain Aging
and in Alzheimer’s Disease. Proc. Natl. Acad. Sci. U. S. A. 107, 14164-14169 (2010).
Liu, D. et al. Screening of Immunophilin Ligands by Quantitative Analysis of Neurofilament Expression and Neurite
Outgrowth in Cultured Neurons and Cells. J. Neurosci. Methods 163, 310-320 (2007).
Liu, R. et al. Pinocembrin Protects against B-Amyloid-Induced Toxicity in Neurons through Inhibiting Receptor
for Advanced Glycation End Products (Rage)-Independent Signaling Pathways and Regulating MitochondrionMediated Apoptosis. BMC Med. 10, 105 (2012).
Lodge, A.P., Langmead, C.J., Daniel, G., Anderson, G.W. & Werry, T.D. Performance of Mouse Neural Stem Cells
as a Screening Reagent: Characterization of Pac1 Activity in Medium-Throughput Functional Assays. J. Biomol.
Screen. 15, 159-168 (2010).
Lotharius, J. et al. Progressive Degeneration of Human Mesencephalic Neuron-Derived Cells Triggered by
Dopamine-Dependent Oxidative Stress Is Dependent on the Mixed-Lineage Kinase Pathway. J. Neurosci. 25,
6329-6342 (2005).
Low, J. et al. Knockdown of Cancer Testis Antigens Modulates Neural Stem Cell Marker Expression in
Glioblastoma Tumor Stem Cells. J. Biomol. Screen. 15, 830-839 (2010).
MacGillavry, H.D. et al. Nfil3 and Camp Response Element-Binding Protein Form a Transcriptional Feedforward
Loop That Controls Neuronal Regeneration-Associated Gene Expression. J. Neurosci. 29, 15542-15550 (2009).
Mallon, R. et al. Antitumor Efficacy Profile of Pki-402, a Dual Phosphatidylinositol 3-Kinase/Mammalian Target of
Rapamycin Inhibitor. Mol. Cancer Ther. 9, 976-984 (2010).
Matsui, H. et al. Pink1 and Parkin Complementarily Protect Dopaminergic Neurons in Vertebrates. Hum. Mol.
Genet. 22, 2423-2434 (2013).
McNeish, J. et al. High-Throughput Screening in Embryonic Stem Cell-Derived Neurons Identifies Potentiators
of α-Amino-3-Hydroxyl-5-Methyl-4-Isoxazolepropionate-Type Glutamate Receptors. J. Biol. Chem. 285, 1720917217 (2010).
Moore, D.L. et al. Klf Family Members Regulate Intrinsic Axon Regeneration Ability. Science 326, 298-301 (2009).
Nakamura, Y., Lee, S., Haddox, C.L., Weaver, E.J. & Lemmon, V.P. The Role of the Cytoplasmic Domain of the L1
Cell Adhesion Molecule in Brain Development. J. Comp. Neurol. 518, 1113-1132 (2010).
Nguyen, L. et al. Quantifying Amyloid Beta (Abeta)-Mediated Changes in Neuronal Morphology in Primary
Cultures: Implications for Phenotypic Screening. J. Biomol. Screen. 17, 835-842 (2012).
Noël, G., Stevenson, S. & Moukhles, H. A High Throughput Screen Identifies Chemical Modulators of the LamininInduced Clustering of Dystroglycan and Aquaporin-4 in Primary Astrocytes. PLoS One 6 (2011).
Oliva, A.A., Jr, Atkins, C.M., Copenagle, L. & Banker, G.A. Activated C-Jun N-Terminal Kinase Is Required for Axon
Formation. J. Neurosci. 26, 9462-9470 (2006).
Papkovskaia, T.D. et al. G2019s Leucine-Rich Repeat Kinase 2 Causes Uncoupling Protein-Mediated
Mitochondrial Depolarization. Hum. Mol. Genet. 21, 4201-4213 (2012).
Pease, M.E. et al. Effect of Cntf on Retinal Ganglion Cell Survival in Experimental Glaucoma. Invest. Ophthalmol.
Vis. Sci. 50, 2194-2200 (2009).
Pescini Gobert, R. et al. Convergent Functional Genomics of Oligodendrocyte Differentiation Identifies Multiple
Autoinhibitory Signaling Circuits. Mol. Cell. Biol. 29, 1538-1553 (2009).
Qian, M.D. et al. Novel Agonist Monoclonal Antibodies Activate Trkb Receptors and Demonstrate Potent
Neurotrophic Activities. J. Neurosci. 26, 9394-9403 (2006).
Radio, N.M., Breier, J.M., Shafer, T.J. & Mundy, W.R. Assessment of Chemical Effects on Neurite Outgrowth in
Pc12 Cells Using High Content Screening. Toxicol. Sci. 105, 106-118 (2008).
Radio, N.M., Breier, J.M., Shafer, T.J. & Mundy, W.R. Assessment of Chemical Effects on Neurite Outgrowth in
PC12 Cells Using High Content Screening. Cold Spring Harb. Protoc. 2010, pdb.tab2top84- (2010).
Radio, N.M., Freudenrich, T.M., Robinette, B.L., Crofton, K.M. & Mundy, W.R. Comparison of PC12 and Cerebellar
Granule Cell Cultures for Evaluating Neurite Outgrowth Using High Content Analysis. Neurotoxicol. Teratol. 32, 2535 (2010).
Richards, G.R., Millard, R.M., Leveridge, M., Kerby, J. & Simpson, P.B. Quantitative Assays of Chemotaxis and
Chemokinesis for Human Neural Cells. Assay Drug Dev. Technol. 2, 465-472 (2004).
Rigamonti, D. et al. Loss of Huntingtin Function Complemented by Small Molecules Acting as Repressor Element
1/Neuron Restrictive Silencer Element Silencer Modulators. J. Biol. Chem. 282, 24554-24562 (2007).
Robinette, B.L., Harrill, J.A., Mundy, W.R. & Shafer, T.J. In Vitro Assessment of Developmental Neurotoxicity: Use
of Microelectrode Arrays to Measure Functional Changes in Neuronal Network Ontogeny1. Front Neuroeng. 4
(2011).
Roy Chowdhury, S.K. et al. Impaired Adenosine Monophosphate-Activated Protein Kinase Signalling in Dorsal
Root Ganglia Neurons Is Linked to Mitochondrial Dysfunction and Peripheral Neuropathy in Diabetes. Brain 135,
1751-1766 (2012).
Ruan, B. et al. Binding of Rapamycin Analogs to Calcium Channels and Fkbp52 Contributes to Their
Neuroprotective Activities. Proc. Natl. Acad. Sci. U. S. A. 105, 33-38 (2008).
Ruscher, K. et al. The Sigma-1 Receptor Enhances Brain Plasticity and Functional Recovery after Experimental
Stroke. Brain 134, 732-746 (2011).
Saito, S., Honma, K., Kita-Matsuo, H., Ochiya, T. & Kato, K. Gene Expression Profiling of Cerebellar Development
with High-Throughput Functional Analysis. Physiol. Genomics 22, 8-13 (2005).
Santos, A.R.C. et al. B1 Integrin-Focal Adhesion Kinase (Fak) Signaling Modulates Retinal Ganglion Cell (Rgc)
Survival. PLoS One 7 (2012).
Sarkar, S. et al. Complex Inhibitory Effects of Nitric Oxide on Autophagy. Mol. Cell 43, 19-32 (2011).
Scannevin, R.H. et al. Fumarates Promote Cytoprotection of Central Nervous System Cells against Oxidative
Stress Via the Nuclear Factor (Erythroid-Derived 2)-Like 2 Pathway. J. Pharmacol. Exp. Ther. 341, 274-284 (2012).
Schildknecht, S. et al. Neuroprotection by Minocycline Caused by Direct and Specific Scavenging of Peroxynitrite.
J. Biol. Chem. 286, 4991-5002 (2011).
Schmidt, F. et al. Identification of Vhy/Dusp15 as a Regulator of Oligodendrocyte Differentiation through a
Systematic Genomics Approach. PLoS One 7 (2012).
Simpson, P.B. et al. Retinoic Acid Evoked-Differentiation of Neuroblastoma Cells Predominates over Growth
Factor Stimulation: An Automated Image Capture and Quantitation Approach to Neuritogenesis. Anal. Biochem.
298, 163-169 (2001).
Stiegler, N.V., Krug, A.K., Matt, F. & Leist, M. Assessment of Chemical-Induced Impairment of Human Neurite
Outgrowth by Multiparametric Live Cell Imaging in High-Density Cultures. Toxicol. Sci. 121, 73-87 (2011).
Strum, J.C. et al. 13th International Symposium on Neural Regeneration (Isnr) Microrna 132 Regulates Nutritional
Stress-Induced Chemokine Production through Repression of Sirt1. Neurorehabil. Neural Repair 23, 954-1000
(2009).
Tan, C.W., Chan, Y.F., Sim, K.M., Tan, E.L. & Poh, C.L. Inhibition of Enterovirus 71 (Ev-71) Infections by a Novel
Antiviral Peptide Derived from Ev-71 Capsid Protein Vp1. PLoS One 7 (2012).
Theus, M.H., Ricard, J., Bethea, J.R. & Liebl, D.J. Ephb3 Inhibits the Expansion of Neural Progenitor Cells in
the Svz by Regulating p53 During Homeostasis and Following Traumatic Brain Injury. Stem Cells 28, 1231-1242
(2010).
Toops, K.A., Berlinicke, C., Zack, D.J. & Nickells, R.W. Hydrocortisone Stimulates Neurite Outgrowth from Mouse
Retinal Explants by Modulating Macroglial Activity. Invest. Ophthalmol. Vis. Sci. 53, 2046-2061 (2012).
Torper, O. et al. Generation of Induced Neurons Via Direct Conversion in Vivo. Proc. Natl. Acad. Sci. U. S. A. 110,
7038-7043 (2013).
Underwood, B.R. et al. Antioxidants Can Inhibit Basal Autophagy and Enhance Neurodegeneration in Models of
Polyglutamine Disease. Hum. Mol. Genet. 19, 3413-3429 (2010).
Usher, L.C. et al. A Chemical Screen Identifies Novel Compounds That Overcome Glial-Mediated Inhibition of
Neuronal Regeneration. J. Neurosci. 30, 4693-4706 (2010).
Vergara, M.N., Gutierrez, C. & Canto-Soler, M.V. New Ex-Ovo Electroporation Technique Offers High Transfection
Efficiency for Primary Retinal Cell Cultures. Invest. Ophthalmol. Vis. Sci. 53, 1121- (2012).
von Schack, D. et al. Dynamic Changes in the Microrna Expression Profile Reveal Multiple Regulatory Mechanisms
in the Spinal Nerve Ligation Model of Neuropathic Pain. PLoS One 6 (2011).
Wheeler, H.E. et al. Integration of Cell Line and Clinical Trial Genome-Wide Analyses Supports a Polygenic
Architecture of Paclitaxel-Induced Sensory Peripheral Neuropathy. Clin. Cancer Res. 19, 491-499 (2013).
Wickstrom, M. et al. The Novel Melphalan Prodrug J1 Inhibits Neuroblastoma Growth in Vitro and in Vivo. Mol.
Cancer Ther. 6, 2409-2417 (2007).
Williams, A.L., Dandepally, S.R., Gilyazova, N., Witherspoon, S.M. & Ibeanu, G. Microwave-Assisted Synthesis of
4-Chloro-N-(Naphthalen-1-Ylmethyl)-5-(3-(Piperazin-1-Yl)Phenoxy)Thiophene-2-Sulfo Namide (B-355252): A New
Potentiator of Nerve Growth Factor (Ngf)-Induced Neurite Outgrowth. Tetrahedron 66, 9577-9581 (2010).
Williams, G. et al. Ganglioside Inhibition of Neurite Outgrowth Requires Nogo Receptor Function: Identification of
Interaction Sites and Development of Novel Antagonists. J. Biol. Chem. 283, 16641-16652 (2008).
Wong, H.K. et al. Blocking Acid-Sensing Ion Channel 1 Alleviates Huntington’s Disease Pathology Via an UbiquitinProteasome System-Dependent Mechanism. Hum. Mol. Genet. 17, 3223-3235 (2008).
Wood-Kaczmar, A. et al. Pink1 Is Necessary for Long Term Survival and Mitochondrial Function in Human
Dopaminergic Neurons. PLoS One 3 (2008).
Wright, K.T., Griffiths, G.J. & Johnson, W.E.B. A Comparison of High-Content Screening Versus Manual Analysis
to Assay the Effects of Mesenchymal Stem Cell-Conditioned Medium on Neurite Outgrowth in Vitro. J. Biomol.
Screen. 15, 576-582 (2010).
Yeyeodu, S.T., Witherspoon, S.M., Gilyazova, N. & Ibeanu, G.C. A Rapid, Inexpensive High Throughput Screen
Method for Neurite Outgrowth. Curr. Chem. Genomics 4, 74-83 (2010).
Yin, Y. et al. Oncomodulin Links Inflammation to Optic Nerve Regeneration. Proc. Natl. Acad. Sci. U. S. A. 106,
19587-19592 (2009).
Zago, W. et al. Neutralization of Soluble, Synaptotoxic Amyloid β Species by Antibodies Is Epitope Specific. J.
Neurosci. 32, 2696-2702 (2012).
Zhang, L. et al. Small Molecule Regulators of Autophagy Identified by an Image-Based High-Throughput Screen.
Proc. Natl. Acad. Sci. U. S. A. 104, 19023-19028 (2007).
Zheng, J. et al. Latanoprost Promotes Neurite Outgrowth in Differentiated Rgc-5 Cells Via the Pi3k-Akt-Mtor
Signaling Pathway. Cell. Mol. Neurobiol. 31, 597-604 (2011).
Zhou, Y. et al. The Zfx Gene Is Expressed in Human Gliomas and Is Important in the Proliferation and Apoptosis
of the Human Malignant Glioma Cell Line U251. J. Exp. Clin. Cancer Res. 30, 114 (2011).
thermoscientific.com/highcontent
©2013 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries. Specifications, terms and
pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details.
USA +1 800 432 4091 [email protected]
Asia +81 3 5826 1659 [email protected]
Europe +32 (0)53 85 71 84 [email protected]
C-AC_DNT0913