- 1. CellPlayer NeuroTrack AssayAssay Design SoftwareDemonstrated
compatibilityIntegrated ,User-friendly with primary neurons,
IncuCyteTM ZOOMneuronal cell lines, and Primary Neutons from
neurons derived from Neuromics iPSCs.QuantitativeNeurite
DynamicsAdvantages Label-free Quantitative, kinetic data 10x or 20x
objectives Flexible algorithm High-definition images Time-lapse
movies Internal Expertise w .essenbi osci ence.comww
2. Tracking Neurite DynamicsNeurite OutgrowthFukata et al.,
Neuroscience Research, Vol. 43, Issue 4, August 2002, Pages 305315w
.essenbi osci ence.com ww 3. Tracking Neurite DynamicsFundamental
Role InNeurite Outgrowth Embryonic development Neuronal
differentiation Nervous system function Neuropathologicaldisorders
Neuronal injury andregeneration Neurotoxicity Fukata et al.,
Neuroscience Research, Vol. 43, Issue 4, August 2002, Pages 305315
w .essenbi osci ence.comww 4. Neurite Outgrowth Analysis:High
Content Imaging ApproachFix cells- Single time point-
Paraformaldehyde fixation -risk loss of fine neuritesAntibody
Labeling (immunofluorescence)- Protocols require a few hours at
minimum- Multiple antibodies Image Acquisition and Analysis - High
Content Imager and software w .essenbi osci ence.comww 5. Neurite
Outgrowth Analysis:High Content Imaging ApproachFix cells- Single
time point- Paraformaldehyde fixation -risk loss of fine
neuritesAntibody Labeling (immunofluorescence)- Protocols require a
few hours at minimum- Multiple antibodies Image Acquisition and
Analysis - High Content Imager and software Labor intensive,
complex, results in data from a single time point. w .essenbi osci
ence.comww 6. NeuroTrack Assay Protocol Cortical Neurons
Hippocampal NeuronsNeuro-2a Cells iCell NeuronsSelect Cells: We
recommend Techno Plastic Products (TPP)Plate Cells: tissue culture
plates for optimal clarity. Change media 18-24hrs post cell
plating. Apply testMedia Change: agents (compounds, growth
factors). Place vessels in IncuCyte Zoom and image at userImage
cells: defined intervals.w .essenbi osci ence.com ww 7. Measuring
Neurite Dynamics withNeuroTrack Non-labeled E18 rat cortical
neurons plated on poly-D-lysineHD PhaseSegmentationT=24hrsT=72hrs
T=120hrs w .essenbi osci ence.comww 8. NeuroTrack is compatible
with primary neurons in phasew .essenbi osci ence.com ww 9.
NeuroTrack Quantifies Neurite Dynamics inReal-Time w .essenbi osci
ence.comww 10. Measuring Neurite Dynamics with NeuroTrack Time
lapse series of images and masksQuantitative dataTime lapse
moviesNeurite Length 15016k cells/wellNeurite length (mm/mm2
)Neurite Length/Cell Body ClusterNeurite length (mm/cell body
cluster)12k cells/well0.8Branch Points1008k cells/well 4k
cells/well4k cells/well0.64000 8k cells/well 16k cells/wellBranch
Points (1/mm2 ) 12k cells/well5012k cells/well0.43000 16k
cells/well8k cells/well 4k cells/well0.22000 0 0 24 48
7296120144Time post plating (hours)0.010000 24 48 7296 120 144 Time
post plating (hours) 0 0 24 48 72 96 120 144Time post plating
(hours) w .essenbi osci ence.comww 11. Assay Validation: NeuroTrack
vs. Endpoint AssayAB Data points represent mean SD, n=30NeuroTrack
quantitation of living neurites in HD phase iscomparable to the
quantitation of fixed and stainedA) NeuroTrack phase image with
maskneurites in a high content imager. B) -tubulin staining in
fixed cells w .essenbi osci ence.comww 12. Low Intra Assay
Variability Non-labeled E18 rat cortical neurons plated on
poly-D-lysine96-well Plate View Data points represent mean SD, n=96
w .essenbi osci ence.comww 13. E18 Rat Cortical Neurons:NeuroTrack
Assay Optimizationw .essenbi osci ence.com ww 14. Cytochalasin D
treatment of E18 cortical neurons highlights the importance of a
kinetic read-out Cytochalasin D depolymerizes the actin
cytoskeleton.Cytochalasin D Treatment: Neurite LengthNeurite length
(mm/cell body cluster)It has been shown that treatment of neurons
with high0.25Cytochalasin Dconcentrations of Cytochalasin D results
in the rapidconcentrationdevelopment of multiple axon-like
structures. * 0.201 M0.3 M However, a NeuroTrack time course
reveals that these0.15Vehiclestructures are transient due to
neurite disintegration and cell 0.100.1 Mdeath.0.05 In contrast,
low concentrations of Cytochalasin D inhibit overallmean SD, n=6, 9
images /well0.00neurite outgrowth. 0 24 48 7296 120 144Time post
plating (hours) w .essenbi osci ence.comww 15. Cytochalasin D
treatment of E18 cortical neurons highlights the importance of a
kinetic read-out Cytochalasin D depolymerizes the actin
cytoskeleton. Cytochalasin D Treatment:Neurite Length Neurite
length (mm/cell body cluster) It has been shown that treatment of
neurons with high 0.25Cytochalasin Dconcentrations of Cytochalasin
D results in the rapid concentrationdevelopment of multiple
axon-like structures. *0.201 M 0.3 M However, a NeuroTrack time
course reveals that these 0.15Vehiclestructures are transient due
to neurite disintegration and cell0.10 0.1 Mdeath. 0.05 In
contrast, low concentrations of Cytochalasin D inhibit overall mean
SD, n=6, 9 images /well 0.00neurite outgrowth.0 24 48 7296 120 144
Time post plating (hours)A B A: Cortical neurons treated with
vehicle (T=66hrs) B: Cortical neurons treated with1 M Cyto D
(T=66hrs) *Bradke and Dotti, Science, 1999 Data points represent
mean SD, n=6 w .essenbi osci ence.comww 16. Cytochalasin D
treatment of E18 cortical neurons highlights the importance of a
kinetic read-outCytochalasin D depolymerizes the actin
cytoskeleton. Cytochalasin D Treatment: Neurite LengthNeurite
length (mm/cell body cluster)It has been shown that treatment of
neurons with high0.25Cytochalasin D concentrations of Cytochalasin
D results in the rapid concentration development of multiple
axon-like structures. *0.201 M0.3 MHowever, a NeuroTrack time
course reveals that these 0.15Vehicle structures are transient due
to neurite disintegration and cell0.100.1 M death.0.05In contrast,
low concentrations of Cytochalasin D inhibit overall mean SD, n=6,
9 images /well0.00 neurite outgrowth. 0 24 48 7296 120 144A B Time
post plating (hours) A B BA: Cortical neurons treatedwith vehicle
(T=66hrs)B: Cortical neuronstreated with1 M Cyto D(T=66hrs)*Bradke
and Dotti, Science, 1999 Data points represent mean SD, n=6w
.essenbi osci ence.com ww 17. Measure Neurite Dynamics
andCytotoxicity Measure cytotoxicity and neurite outgrowth
kinetically in thesame well.YoPro-3 (Life Technologies) is a cell
impermeant cyanine dimernucleic acid stain that binds dsDNA.
Apoptosis and necrosis result ina loss of membrane integrity.
YoPro-3 stains cell nuclei only whencells have lost membrane
integrity, viable cells remain unstained.YoPro-3+ cytotoxic
compoundWe have optimized the use of YoPro-3 for use in
monitoringcytotoxicity kinetically in primary cortical neurons.
Control0.1 M R0-31-8220 1 M R0-31-8220w .essenbi osci ence.com ww
18. Measure Neurite Dynamics and Cytotoxicity Measure cytotoxicity
and neurite outgrowth kinetically in thesame well.YoPro-3 (Life
Technologies) is a cell impermeant cyanine dimernucleic acid stain
that binds dsDNA. Apoptosis and necrosis result ina loss of
membrane integrity. YoPro-3 stains cell nuclei only whencells have
lost membrane integrity, viable cells remain unstained.YoPro-3+
cytotoxic compoundWe have optimized the use of YoPro-3 for use in
monitoringcytotoxicity kinetically in primary cortical
neurons.Control 0.1 M R0-31-8220 1 M R0-31-8220w .essenbi osci
ence.com ww 19. Neurite Outgrowth + Cytotoxicity Measuring
cytotoxicity and neurite length in response to PKC inhibition: 24
hours post plating, E18 rat cortical neurons were treated with
different concentrations of the PKC inhibitor, Ro-31-8220, in the
presence of Yo-Pro3 .Neurite Length/Cell Body ClusterNeurite length
(mm/cell body cluster)0.5 Vehicle 0.004 M Ro-31-82200.4 0.02 M
Ro-31-82200.30.1 M Ro-31-8220 0.5 M Ro-31-82200.21 M
Ro-31-82200.10.00 2448 72 96120 144 Time post plating (hours) Data
points represent mean SD, n=6, 9 images/wellw .essenbi osci
ence.com ww 20. Neurite Outgrowth + Cytotoxicity Measuring
cytotoxicity and neurite length in response to PKC inhibition: 24
hours post plating, E18 rat cortical neurons were treated with
different concentrations of the PKC inhibitor, Ro-31-8220, in the
presence of Yo-Pro3 .Neurite Length/Cell Body Cluster Cell
DeathNeurite length (mm/cell body cluster) YoPro-3 Red Object
Count/mm20.5 300 Vehicle 0.004 M Ro-31-82200.4 1 M Ro-31-8220 0.02
M Ro-31-8220 0.5 M Ro-31-82202000.30.1 M Ro-31-82200.1 M Ro-31-8220
0.5 M Ro-31-82200.02 M Ro-31-82200.21 M Ro-31-8220100 0.004 M
Ro-31-8220Vehicle0.10.000 2448 72 96120 144 0 24487296120 144 Time
post plating (hours) Time post plating (hours) Data points
represent mean SD, n=6, 9 images/wellw .essenbi osci ence.com ww
21. CellPlayer NeuroTrack Assay The HD Phase optics, integrated
software algorithm and live-cellLabel-free:imaging obviates the
need to fix and label cells. Time lapse measurement of neurite
dynamics under physiologicalKinetic: conditions. Images can be
assembled into time-lapse movies.Compatible with multiple Validated
with rodent primary neurons, iPSC derived neurons andneuronal cell
types: Neuro-2A cells. Automated data acquisition and integrated
metric calculationsEasy to use software:provide convenient access
to complex data. Multiplex: Monitor a fluorescent label as well as
neurite dynamics. w .essenbi osci ence.comww