Applications

Neurite Analysis

Overview

What is neurite outgrowth?

During development, neurons extend numerous processes that differentiate into dendrites and axons. These processes (termed neurites) are critical for communication between neurons. Characterization of neurite outgrowth, maturation, and the disruption of neurite networks is key to the study of neuropathological disorders (e.g. neurodegeneration), neuronal injury/regeneration and screening for neurotoxicity.

Measurements of neurite outgrowth are commonly made following antibody labelling or staining of the neuronal cultures. These methods require cell fixing and include multiple, manual wash/preparation steps that ultimately kill the neurons and may disrupt delicate neurite structures. Measurements of neurite length are usually made at a single time point, or at best a limited series of time points, as cultures need to be shuttled into and out of the incubator for analysis. Neurite outgrowth assays are often short term (24 or 48 h) and lack information associated with longer term neuronal biology such as the development and disruption of mature neural networks.

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INCUCYTE® NEURITE ANALYSIS

Real-time neurite analysis inside your incubator

IncuCyte® Neurite Analysis Assays enable automated, continuous analysis of neurite outgrowth and neural network stability - all inside your cell culture incubator. Quantify neurite length and branch points in your choice of neurons (e.g. primary, iPSC-derived, immortalized neuronal cell lines) in monoculture or in co-culture with astrocytes.

  • Screen for neurotoxicity
  • Investigate neuroprotective treatments
  • Study pathways involved in neurodegeneration
  • Measure neuronal differentiation in real time

1. Label-free analysis of neurons
in monoculture

Monitor neurite outgrowth in live neuronal monocultures using IncuCyte NeuroTrack™ analysis software. Fully automated, long-term label-free analysis.

Human iPSC-derived neurons (iCell Neurons, CDI) in monoculture

Human iPSC-derived neurons (iCell Neurons, CDI) in monoculture.

2. Fluorescence analysis of neurons in co-culture with astrocytes

Measure changes in neurite length and branch points in mixed cultures of neurons and astrocytes using fluorescent neuronal labelling reagents and IncuCyte analysis software.

Primary rat forebrain neurons labeled with the IncuCyte® NeuroLight Red reagent in co-culture with rat astrocytes

Primary rat forebrain neurons labeled with the IncuCyte® NeuroLight Red reagent in co-culture with rat astrocytes.

Assay Concept

IncuCyte® Neurite Analysis Concept

  1. Measure neurite dynamics automatically and in real time from living cultures over days/weeks using the IncuCyte® Live-Cell Analysis system and NeuroTrack software.
  2. Monitor changes in neurite length and branch points by label-free analysis of neurons in monoculture OR use neuron specific live-cell labeling reagents to quantify neurite dynamics in mixed cultures of neurons and astrocytes
  3. Multiplex neurite analysis with cell viability readouts by combining with mix-and-read reagents for contiuous monitoring of cytotoxicity and apoptosis - distinguish treatments that affect one or more aspects of neuronal cell health.

Key Advantages

Key Advantages of IncuCyte® Neurite Analysis

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Visualize and quantify neurite outgrowth and disruption in real time

  • Visualize and analyze long-term changes in neurite length and branch points with the IncuCyte® Live-Cell Analysis System and NeuroTrack software.
  • Study label-free neurons in monoculture, or use IncuCyte® NeuroLight reagents or NeuroPrime Cell Kits for selective analysis of neurite dynamics in co-cultures.

Continuous measures of neurite dynamics

Continuous measures of neurite dynamics. IncuCyte® NeuroTrack analysis software automatically analyzes images to provide measures of neurite length, branch points and neuronal cell body clusters.


Automatically generate time courses and reveal concentration-dependent responses

  • Set up, walk away and receive unbiased, automated analyses – plates do not need to be removed from the incubator for measurement
  • Run experiments in up to six 96- or 384-well plates at one time

neurite_automatically-generate

Measure treatment effects automatically and non-invasively. IncuCyte neurite analysis assays allow every well of a 96- or 384-well plate to be imaged and analyzed automatically to provide a microplate readout of neurite dynamics over time (A). Time-courses reveal concentration-dependent treatment effects (B). Transform data into concentration-response curves to compare pharmacology (C).

Create relevant models of neurodegeneration and neurotoxicity

  • Make continuous measurements form relevant neuronal models in highly flexible, robust and reproducible assay formats suitable for screening and profiling

Create relevant in vitro models of neurodegeneration and neurotoxicity

Create relevant in vitro models of neurodegeneration and neurotoxicity - screen for neuroprotective treatments. Concentration dependent neurotoxic effects of 6-hydroxydopamine (6-OHDA) measured in human iPSC-derived dopaminergic neurons (Dopa.4U, Axiogenesis) in a functional model of Parkinson’s disease. Treatment with bone derived neural factor (BDNF) promotes neurite formation but provided little or no protection against the effects of 6-OHDA 


Real time live cell protocols, no fixing, no staining no antibody labeling

Neuronal monocultures—label-free protocol overview

Neuronal monocultures label-free protocol overview

Neuronal co-cultures—fluorescence protocol overview

Neuronal co-cultures fluorescence protocol overview

View the label-free neurite analysis protocol

View the fluorescence neurite analysis protocol


Multiplex neurite analysis with cell health readouts for additional insight

  • Use IncuCyte® mix-and-read reagents for continuous monitoring of cytotoxicity and apoptosis in neuronal cultures
  • Simultaneous measurements of neurite dynamics and neuronal cell health

IncuCyte® Cytotox and Annexin V reagents

Use IncuCyte Cytotox and Annexin V reagents to positively mark and quantify apoptotic or cytotoxic cells

Use IncuCyte® Cytotox and Annexin V reagents to positively mark and quantify apoptotic or cytotoxic cells. (Left) IncuCyte® Cytotox Red reagent labels dead rat forebrain neurons treated with 333 μM glutamate. (Right) IncuCyte® Annexin V Green reagent identifies apoptotic cells in a rat forebrain neuron and astrocyte co-culture in the presence and absence of 300 μM glutamate. Neurons are selectively labeled with the IncuCyte® NeuroLight Red reagent. Multiplexed measurements of cell viability and neurite dynamics can be made in real time.

Measure time-courses and concentration-dependent effects on neurite dynamics and cell viability

Measure time-courses and concentration-dependent effects on neurite dynamics and cell viability.  Glutamate excitotoxicity in rat forebrain neurons cultured in the presence of astrocytes: (left) neurite network length is disrupted in a concentration dependent manner, (center) measurements apoptosis reveal early effects on cell health (right) concentration-response curves.

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Ordering Info

Ordering Information

APPLICATION Type PRODUCTS QUANTITY CAT. NO.
Neurite analysis
Measure neurite dynamics in monocultures and co-cultures with astrocytes.
Reagents IncuCyte® NeuroLight™ Red 0.45 mL 4584
IncuCyte® NeuroPrime™ Cell Kit 1 Kit 4585
Software IncuCyte® NeuroTrack™ Software   9600-0010
Apoptosis
Detect and quantify apoptotic cells in real time.
Reagents IncuCyte® Annexin V Red for Apoptosis 1 vial, 100 tests 4641
IncuCyte® Annexin V Green for Apoptosis 1 vial, 100 tests 4642
Caspase-3/7 20 μL 4440
Cytotoxicity
Detect and count non-viable cells in real time.
Reagents IncuCyte® Cytotox Red 5 μL x 5 4632
IncuCyte® Cytotox Green 5 μL x 5 4633
Cell lines
Cryopreserved neuronal cells and astrocytes.
Neuronal
Cells
Neuro-2a NucLight™ Green Cells 2 x 106 cells/vial 4511
Neuro-2a NucLight™ Red Cells 2 x 106 cells/vial 4512
NeuroPrime™ rAstrocytes 2 x 106 cells/vial 4586
NeuroPrime™ rForebrain Neurons 2 x 106 cells/vial 4587

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