CellPlayer NeuroTrack ™ Assay  Assay Design                                           SoftwareDemonstrated compatibility  ...
Tracking Neurite Dynamics                                                          Neurite OutgrowthFukata et al., Neurosc...
Tracking Neurite Dynamics    Fundamental Role In                                                Neurite Outgrowth•   Embry...
Neurite Outgrowth Analysis:                    High Content Imaging ApproachFix cells- Single time point- Paraformaldehyde...
Neurite Outgrowth Analysis:                    High Content Imaging ApproachFix cells- Single time point- Paraformaldehyde...
NeuroTrack ™ Assay Protocol                 Cortical Neurons   Hippocampal Neurons      Neuro-2a Cells   iCell Neurons®Sel...
Measuring Neurite Dynamics with                        NeuroTrack ™                   Non-labeled E18 rat cortical neurons...
NeuroTrack is compatible with primary         neurons in phase              w .essenbi osci ence.com             ww
NeuroTrack ™ Quantifies Neurite Dynamics in                Real-Time                           w .essenbi osci ence.com   ...
Measuring Neurite Dynamics with                                                                 NeuroTrack ™              ...
Assay Validation:             NeuroTrack ™ vs. Endpoint Assay                                                             ...
Low Intra Assay Variability Non-labeled E18 rat cortical neurons plated on poly-D-lysine                                  ...
E18 Rat Cortical Neurons:  NeuroTrack ™ Assay Optimization            w .essenbi osci ence.com           ww
Cytochalasin D treatment of E18 cortical neurons highlights the importance of a kinetic read-out•   Cytochalasin D depolym...
Cytochalasin D treatment of E18 cortical neurons highlights the importance of a kinetic read-out•   Cytochalasin D depolym...
Cytochalasin D treatment of E18 cortical neurons highlights the importance of a kinetic read-out•    Cytochalasin D depoly...
Measure Neurite Dynamics and                    Cytotoxicity• Measure cytotoxicity and neurite outgrowth kinetically in th...
Measure Neurite Dynamics and                   Cytotoxicity• Measure cytotoxicity and neurite outgrowth kinetically in the...
Neurite Outgrowth + Cytotoxicity                                                                                          ...
Neurite Outgrowth + Cytotoxicity                                                                                          ...
CellPlayer NeuroTrack™ Assay                           • The HD Phase optics, integrated software algorithm and live-cell ...
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Essen bioscience neuromics 9_17_12

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Providing tools that insure excellent Cell Based Assays is a cornerstone of our business strategy. Lauren McGillicuddy and her team at Essen Bioscience have been using our E18 Primary Rat Cortical Neurons to develop NeuroTrakTM assays enabling kinetic quantification of neurite dynamics (initiation, branching, extension, retraction). NeuroTrack is one of several CellPlayerTM assays that can be run in IncuCyte ZoomTM.

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Essen bioscience neuromics 9_17_12

  1. 1. CellPlayer NeuroTrack ™ Assay Assay Design SoftwareDemonstrated compatibility Integrated ,User-friendly with primary neurons, IncuCyteTM ZOOM neuronal cell lines, and Primary Neutons from neurons derived from Neuromics iPSCs. Quantitative Neurite Dynamics Advantages • Label-free • Quantitative, kinetic data • 10x or 20x objectives • Flexible algorithm • High-definition images • Time-lapse movies • Internal Expertise w .essenbi osci ence.com ww
  2. 2. Tracking Neurite Dynamics Neurite OutgrowthFukata et al., Neuroscience Research, Vol. 43, Issue 4, August 2002, Pages 305–315 w .essenbi osci ence.com ww
  3. 3. Tracking Neurite Dynamics Fundamental Role In Neurite Outgrowth• Embryonic development• Neuronal differentiation• Nervous system function• Neuropathological disorders• Neuronal injury and regeneration• Neurotoxicity Fukata et al., Neuroscience Research, Vol. 43, Issue 4, August 2002, Pages 305–315 w .essenbi osci ence.com ww
  4. 4. Neurite Outgrowth Analysis: High Content Imaging ApproachFix cells- Single time point- Paraformaldehyde fixation -risk loss of fine neurites Antibody Labeling (immunofluorescence) - Protocols require a few hours at minimum - Multiple antibodies Image Acquisition and Analysis - High Content Imager and software w .essenbi osci ence.com ww
  5. 5. Neurite Outgrowth Analysis: High Content Imaging ApproachFix cells- Single time point- Paraformaldehyde fixation -risk loss of fine neurites Antibody 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.com ww
  6. 6. NeuroTrack ™ Assay Protocol Cortical Neurons Hippocampal Neurons Neuro-2a Cells iCell Neurons®Select 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. 7. Measuring Neurite Dynamics with NeuroTrack ™ Non-labeled E18 rat cortical neurons plated on poly-D-lysineHD PhaseSegmentation T=24hrs T=72hrs T=120hrs w .essenbi osci ence.com ww
  8. 8. NeuroTrack is compatible with primary neurons in phase w .essenbi osci ence.com ww
  9. 9. NeuroTrack ™ Quantifies Neurite Dynamics in Real-Time w .essenbi osci ence.com ww
  10. 10. Measuring Neurite Dynamics with NeuroTrack ™ Time lapse series of images and masks Quantitative data Time lapse movies Neurite Length 150 16k cells/well Neurite length (mm/mm2 ) Neurite Length/Cell Body ClusterNeurite length (mm/cell body cluster) 12k cells/well 0.8 Branch Points 100 8k cells/well 4k cells/well 4k cells/well 0.6 4000 8k cells/well 16k cells/well Branch Points (1/mm2 ) 12k cells/well 50 12k cells/well 0.4 3000 16k cells/well 8k cells/well 4k cells/well 0.2 2000 0 0 24 48 72 96 120 144 Time post plating (hours) 0.0 1000 0 24 48 72 96 120 144 Time post plating (hours) 0 0 24 48 72 96 120 144 Time post plating (hours) w .essenbi osci ence.com ww
  11. 11. Assay Validation: NeuroTrack ™ vs. Endpoint Assay A B • Data points represent mean ± SD, n=30NeuroTrack quantitation of living neurites in HD phase is comparable to the quantitation of fixed and stained A) NeuroTrack phase image with mask neurites in a high content imager. B) β-tubulin staining in fixed cells w .essenbi osci ence.com ww
  12. 12. Low Intra Assay Variability Non-labeled E18 rat cortical neurons plated on poly-D-lysine 96-well Plate View • Data points represent mean ± SD, n=96 w .essenbi osci ence.com ww
  13. 13. E18 Rat Cortical Neurons: NeuroTrack ™ Assay Optimization w .essenbi osci ence.com ww
  14. 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 Length• Neurite length (mm/cell body cluster) It has been shown that treatment of neurons with high 0.25 Cytochalasin D concentrations of Cytochalasin D results in the rapid concentration development of multiple axon-like structures. * 0.20 1 µM 0.3 µM• However, a NeuroTrack time course reveals that these 0.15 Vehicle structures are transient due to neurite disintegration and cell 0.10 0.1 µM death. 0.05• In contrast, low concentrations of Cytochalasin D inhibit overall mean ± SD, n=6, 9 images /well 0.00 neurite outgrowth. 0 24 48 72 96 120 144 Time post plating (hours) w .essenbi osci ence.com ww
  15. 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 0.25 Cytochalasin D concentrations of Cytochalasin D results in the rapid concentration development of multiple axon-like structures. * 0.20 1 µM 0.3 µM• However, a NeuroTrack time course reveals that these 0.15 Vehicle structures are transient due to neurite disintegration and cell 0.10 0.1 µM death. 0.05• In contrast, low concentrations of Cytochalasin D inhibit overall mean ± SD, n=6, 9 images /well 0.00 neurite outgrowth. 0 24 48 72 96 120 144 Time post plating (hours)A B A: Cortical neurons treated with vehicle (T=66hrs) B: Cortical neurons treated with1 µM Cyto D (T=66hrs) *Bradke and Dotti, Science, 1999 • Data points represent mean ± SD, n=6 w .essenbi osci ence.com ww
  16. 16. 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 0.25 Cytochalasin D concentrations of Cytochalasin D results in the rapid concentration development of multiple axon-like structures. * 0.20 1 µM 0.3 µM• However, a NeuroTrack time course reveals that these 0.15 Vehicle structures are transient due to neurite disintegration and cell 0.10 0.1 µM death. 0.05• In contrast, low concentrations of Cytochalasin D inhibit overall mean ± SD, n=6, 9 images /well 0.00 neurite outgrowth. 0 24 48 72 96 120 144A B Time post plating (hours) A B B A: Cortical neurons treated with vehicle (T=66hrs) B: Cortical neurons treated with1 µM Cyto D (T=66hrs) *Bradke and Dotti, Science, 1999 • Data points represent mean ± SD, n=6 w .essenbi osci ence.com ww
  17. 17. Measure Neurite Dynamics and Cytotoxicity• Measure cytotoxicity and neurite outgrowth kinetically in the same 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 µM R0-31-8220 1 µM R0-31-8220 w .essenbi osci ence.com ww
  18. 18. Measure Neurite Dynamics and Cytotoxicity• Measure cytotoxicity and neurite outgrowth kinetically in the same 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 µM R0-31-8220 1 µM R0-31-8220 w .essenbi osci ence.com ww
  19. 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 µM Ro-31-8220 0.4 0.02 µM Ro-31-8220 0.3 0.1 µM Ro-31-8220 0.5 µM Ro-31-8220 0.2 1 µM Ro-31-8220 0.1 0.0 0 24 48 72 96 120 144 Time post plating (hours) • Data points represent mean ± SD, n=6, 9 images/well w .essenbi osci ence.com ww
  20. 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/mm2 0.5 300 Vehicle 0.004 µM Ro-31-8220 0.4 1 µM Ro-31-8220 0.02 µM Ro-31-8220 0.5 µM Ro-31-8220 200 0.3 0.1 µM Ro-31-8220 0.1 µM Ro-31-8220 0.5 µM Ro-31-8220 0.02 µM Ro-31-8220 0.2 1 µM Ro-31-8220 100 0.004 µM Ro-31-8220 Vehicle 0.1 0.0 0 0 24 48 72 96 120 144 0 24 48 72 96 120 144 Time post plating (hours) Time post plating (hours) • Data points represent mean ± SD, n=6, 9 images/well w .essenbi osci ence.com ww
  21. 21. CellPlayer NeuroTrack™ Assay • The HD Phase optics, integrated software algorithm and live-cell Label-free: imaging obviates the need to fix and label cells. • Time lapse measurement of neurite dynamics under physiological Kinetic: conditions. Images can be assembled into time-lapse movies.Compatible with multiple • Validated with rodent primary neurons, iPSC derived neurons and neuronal cell types: Neuro-2A cells. • Automated data acquisition and integrated metric calculations Easy to use software: provide convenient access to complex data. Multiplex: • Monitor a fluorescent label as well as neurite dynamics. w .essenbi osci ence.com ww

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