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Neurite Outgrowth

Gain valuable insights for interpreting neurobiology, from evaluation of iPSC-derived neurons to analysis of 3D neuron organoids.

Simplify characterization of neurons using neurite outgrowth assay to study neuronal development and degeneration 𝘪𝘯 𝘷𝘪𝘵𝘳𝘰

Neurons create connections via extensions of their cellular body called axons and dendrites, which are commonly referred to as “neurites” or “processes”. This biological phenomenon is referred to as neurite outgrowth and is regulated by complex intracellular signaling events.

Neurite outgrowth is a commonly used assay to study neuronal development and neuronal degeneration in vitro. Development of neurites requires a complex interplay of both extracellular and intracellular signals. The growth of neurites can be stimulated or inhibited by neurotrophic factors. Importantly, the development of neurons can be affected by neurotoxic chemicals.

Understanding the signaling mechanisms driving neurite outgrowth provides valuable insight for interpreting neurotoxic responses and compound screening data and for interpreting factors influencing neural development and regeneration. Inhibition or stimulation of neurite outgrowth is implicated in a broad range of CNS disorders or injuries including stroke, Parkinson’s disease, Alzheimer’s disease, and spinal cord injuries.

Workflow solution for analyzing neurite outgrowth

Neurite outgrowth is assessed by the segmentation and quantification of neuronal processes. These neuronal processes can be imaged using a fluorescence microscope and quantified with manual tracing and counting when throughput is low. However, for samples in a higher-throughput microplate format, an automated imaging system paired with analysis software is a more efficient solution.

 

Neurite Workflow

 

 

The workflow illustrates a simplified process for analyzing neurite outgrowth and highlights systems to help you streamline your research and increase your throughput.

  1. Culture neuronal cells – cells were grown and allowed to form neurite networks in 96- or 384-well microplates.

  2. Treat with compounds – the cells were then exposed to toxic compounds for 48 hours.

  3. Stain for markers – After compound treatment is complete, live cell stains can be added directly to the media. Immunostaining protocols with fluorescently-conjugated antibodies can also be performed post-cell fixation.

  4. Acquire neuronal images – High-content imaging of neurons allows scientists to both characterize and measure changes in neuronal networks such as neurite number, length, and branching, as well as to determine gross or specific toxicity reactions. Acquire images with large field-of-view optics so more cells can be sampled with fewer sites per well, leading to dramatically faster plate acquisition times.

  5. Analyze neuronal network – High content analysis provides a quantitative method to determine effects of positive and negative factors on neurite outgrowth. Use cellular imaging analysis software to run quantitative analysis of the neuronal cell images to characterize several parameters including number of processes per cell, length of neurite outgrowth, branching, and number of cells.

 

 

Neurite outgrowth applications and assays

High-content imaging of neurons allows scientists to both characterize and measure changes in neuronal networks such as neurite number, length, and branching, as well as to determine gross or specific toxicity reactions.

Learn how to capture and quantify neuronal activities quickly and accurately using automated microscopy and high-content analysis software:

  • eBook: Phenotypic Screening with iPSCs

    Phenotypic Screening with iPSCs

    Phenotypic Screening With iPSC-Derived Cardiomyocytes and Neurons

    There is a critical need for more relevant models to identify therapeutic effects and adverse responses to compounds earlier in the drug discovery process. Profiling the effects of environmental hazards in a higher throughput fashion is extremely important. The development of human induced pluripotent stem cells (iPSC) has made it possible to develop new higher throughput screening assays to study compound effects upon iPSC cardiomyocytes and neuronal cultures.

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    Induced pluripotent stem cells (iPSCs)

    Induced pluripotent stem cells (iPSCs)

    High-content imaging using induced pluripotent stem cells (iPSCs) of human origin can be applied to examine neurotrophic, neuroprotective, or neurotoxic effects of pharmaceutical drug candidates or environmental contaminants.

    iPSCs are very useful for neuronal toxicity studies as they exhibit the functionality and behavior of mature neurons, and are also available in large quantities. This biologically relevant cell type paired with high-content imaging and analysis makes neurotoxicity assays valuable for screening lead compounds and potentially reduces pre-clinical development costs and the need for animal experimentation.

  • Neurite outgrowth analysis

    Neurite outgrowth analysis

    The Neurite Outgrowth Application Module for MetaXpress® software is designed for the analysis of neurite outgrowth assays. The module helps standardize results compared to traditional methods. Using a nuclear stain is beneficial to identify cell bodies in some cell types. With the flexibility of MetaXpress software, researchers are able to choose whether or not to use nuclear stains.

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    Neuron 3D model

    Neuron 3D model

    3D neuronal cultures are recognized as more closely recapitulating aspects of the human tissues including the architecture, cell organization, as well as cell-cell and cell-matrix interactions.

    Establishment of physiologically-relevant in vitro models is crucial to further understanding of the mechanisms of neurological diseases as well as targeted drug development. While iPSC-derived neurons show great promise for compound screening and disease modeling, use of three-dimensional (3D) cultures is emerging as a valid approach for neuronal cell based assay development.

  • Neuron morphology

    Neuron morphology

    Neuron morphology (or neuromorphology) refers to the structure and shape of the cells that make up the nervous system. The shapes of nerve cells are highly complex, exemplified by the broad morphological diversity of neuronal processes – axons and dendrites – and their extraordinarily variable and intricate interconnectivity. From monitoring morphological changes during and after development as well as in the progression of disease states to studying the neurotoxic effects of drugs, chemicals and environmental toxicants, the ability to study this sophisticated level of morphological complexity is critical. Furthermore, the relationship between morphology and function is a key area of investigation in a wide variety of neuroscience research areas.

    Neurotoxicity

    Neurotoxicity

    The nervous system is sensitive to the toxic effects of many chemical compounds, environmental agents and certain naturally occurring substances. Neurotoxicity can cause temporary or permanent damage of the brain or peripheral nervous system during pathological processes such as spinal cord injury, stroke, or traumatic brain injury. It is also a major cause of neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease.

  • Scientific poster: Neurotoxicity evaluation using iPSC

    Neurotoxicity evaluation using iPSC

    Multiplexed automated assays for neurotoxicity evaluation using induced pluripotent stem cell-derived neural 3D cell models

    Cell-based phenotypic assays have become an increasingly attractive alternative to traditional in vitro and in vivo testing in pharmaceutical drug development and toxicological safety assessment. The effectiveness of automated imaging assays combined with the organotypic nature of human induced pluripotent stem cell (iPSC)-derived cells opens new opportunities to employ physiologically relevant in vitro model systems to improve screening for new drugs or potential chemical toxicities. In our studies, we used human iPSC-derived neural cultures to test functional and morphological end points for toxicity evaluation in a multi-parametric assay format.

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    Webinar: Quantifying Biological Complexity of iPSC

    Quantifying Biological Complexity of iPSC

    On-demand Webinar: Simplified imaging for quantifying biological complexity of iPSC-derived cardiomyocytes and neurons

    Watch our webinar to learn how our scientists simplify the workflow for multi-parametric biological responses by using the most current automated imaging acquisition and analysis tools.

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Resources of Neurite Outgrowth

Videos & Webinars

Morphological Characterization of 3D Neuronal Networks in a Microfluidic Platform

Morphological Characterization of 3D Neuronal Networks in a Microfluidic Platform

Tunable optogenetic system elucidates the role of beta-catenin signaling dynamics on neural stem cell differentiation

Human iPS Cell-derived Neurons

Human iPS Cell-derived Neurons: A New Physiologically Relevant Model for Drug Discovery Applications for High-Content Screening of Neurotoxicity

Using Electrophysiological Studies to Accelerate Mechanistic in Reception and Transmission

Using Electrophysiological Studies to Accelerate Mechanistic Study in Reception and Transmission

Effects of Amyloid-Beta Proteins on hSlo1.1, a BK Channel, in Xenopus Oocyte Model

Investigations of the Effects of Amyloid-Beta Proteins on hSlo1.1, a BK Channel, in a Xenopus Oocyte Model

Simplified Imaging for Quantifying Biological Complexity of iPSC-Derived Cardiomyocytes and Neurons