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