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.

The workflow illustrates a simplified process for analyzing neurite outgrowth and highlights systems to help you streamline your research and increase your throughput.
- Culture neuronal cells – cells were grown and allowed to form neurite networks in 96- or 384-well microplates.
- Treat with compounds – the cells were then exposed to toxic compounds for 48 hours.
- 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.
- 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.
- 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:
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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
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
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.
- Functional and mechanistic neurotoxicity profiling using 3D neural cultures
- Neuronal development in 3D matrix
- Morphological characterization of 3D neuronal networks in a microfluidic platform
- 3D neurospheroid for high-throughput screening
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.
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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.
Phenotypic screening
eBook: Phenotypic Screening With iPSC-Derived Cardiomyocytes and Neurons
Learn how to use both imaging and calcium oscillation analysis to develop profiles of compounds in iPSC-derived cardiomyocytes such as hERG blockers, ß-adrenergic agonists, and environmental toxins. iPSC-derived neuronal cultures were evaluated with neuromodulators as well as environmental toxins.
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Scientific poster: 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.
Toxicology
Toxicology is the study of adverse effects of natural or man-made chemicals on living organism. It is a growing concern in our world today as we are exposed to more and more chemicals, both in our environment and in the products we use.
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Webinar: 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.
Learn about:
- Functional and cytotoxicity assays using various cell models including iPSC-derived cardiac and neuronal cells
- New analytical tools for collecting and processing multi-parametric readouts
- Methods for adapting and scaling up processes for compound testing
Latest Resources
Resources of Neurite Outgrowth
Application Note
Neurite outgrowth analysis in 3D reconstructed human brain micro-tissues
Neurite outgrowth analysis in 3D reconstructed human brain micro-tissues
Organotypic 3D cultures resemble native 3D tissue architecture and are believed more representative of real tissues than their 2D (monolayer) counterparts, thus providing higher in vivo…
Publications
Benefits of Real-Time Live-Cell Imaging
Benefits of Real-Time Live-Cell Imaging
Three-dimensional (3D) cell-based assays replace a host of tissue, cell, and animal test models but have a reputation for lacking time resolution. For example, traditional, endpoint cell-bas…
Blog
Enabling 3D High-Content Imaging and Analysis on the Organ-on-a-Chip Platform
Enabling 3D High-Content Imaging and Analysis on the Organ-on-a-Chip Platform
Imagine having the ability to mimic the human biological environment for disease modeling and drug screening and doing so in a micro-scale system. With the development of organ-on-a-…
Blog
Tips for running a successful live cell imaging experiment
Tips for running a successful live cell imaging experiment
There have been significant advancements in microscopy and camera technology, as well as advancements in technologies for labeling molecules of interest over the past decade. These…
Publications
Shaping the Future of Organoid Research
Shaping the Future of Organoid Research
Molecular Devices, a provider of innovative life science technology, recently unveiled a brand new, first-of-its-kind Organoid Innovation Center. Situated at the company’s global headquarter…
Publications
Disease Modeling with 3D Cell-Based Assays Using a Novel Flowchip System and High-Content Imaging
Disease Modeling with 3D Cell-Based Assays Using a Novel Flowchip System and High-Content Imaging
There is an increasing interest in using three-dimensional (3D) cell structures for modeling tumors, organs, and tissue to accelerate translational research. We describe here a novel automat…
Publications
How AI is advancing neurodegenerative disease research
How AI is advancing neurodegenerative disease research
In this guest editorial, explore how artificial intelligence is becoming a powerful tool for drug discovery As part of the SelectScience Advances in Drug Discovery Special Feature, guest edi…
Blog
7 tips for optimizing your 3D cell imaging and analysis workflow
7 tips for optimizing your 3D cell imaging and analysis workflow
As the field of cell biology moves toward increasing complexity of assays, 3D cell systems have proven to provide more physiologically relevant information as compared to…
Publications
The workflow game-changer: combined technologies for efficiency
The workflow game-changer: combined technologies for efficiency
Microfluidic flowchip technology combined with three-dimensional (3D) imaging is a powerful duo poised to change the future of drug discovery and development. The integration of combined tec…
Publications
Neuroscience: bridging the gap between cell-based and human research
Neuroscience: bridging the gap between cell-based and human research
Neurological disorders impact up to a billion people around the world and appear to be on the rise [1]. In fact, those affecting the central and peripheral nervous systems – including stroke…
Scientific Poster
Multiplexed automated assays for neurotoxicity evaluation using induced pluripotent stem cell-derived neural 3D cell models
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 assess…
Publications
Revolutionizing early drug discovery for immuno-oncology and neurodegenerative disease modeling
Revolutionizing early drug discovery for immuno-oncology and neurodegenerative disease modeling
The development of 3D cellular models of disease has enabled researchers to better recapitulate in vivo cell environments. As 3D models become more prevalent in the drug discovery industry,…
Presentations
Functional and mechanistic neurotoxicity profiling using human iPSC-derived neural 3D cultures
Functional and mechanistic neurotoxicity profiling using human iPSC-derived neural 3D cultures
Expertise in human iPSC-derived neural and cardiac platforms; Focus on adapting platforms to HTS and producing assay-ready solutions to accelerate drug discovery; Neural platform: mocroBra…
eBook
Phenotypic Screening With iPSC-Derived Cardiomyocytes and Neurons
Phenotypic Screening With iPSC-Derived Cardiomyocytes and Neurons
This new eBook discusses early drug safety testing using HTS iPSC-derived cells with imaging. Download it now!
Application Note
Identify compound-specific effects on neurite outgrowth using ImageXpress Pico system
Identify compound-specific effects on neurite outgrowth using ImageXpress Pico system
Evaluate compound-specific effects on neurite outgrowth using automated imaging
Application Note
Multi-parameter imaging assay for measuring toxicity in a tumor model
Multi-parameter imaging assay for measuring toxicity in a tumor model
There is an increasing interest in using three-dimensional (3D) spheroids for modeling cancer and tissue biology to accelerate translational research. The goal of this study was to develop…
Application Note
Measuring neurite outgrowth with the SpectraMax MiniMax cytometer and MetaMorph software
Measuring neurite outgrowth with the SpectraMax MiniMax cytometer and MetaMorph software
Neurons create connections via extensions of their cellular body called dendrites or “processes”. This biological phenomena is referred to as neurite outgrowth and is regulated by complex…
Application Note
Visualize subcellular vesicles to quantitate autophagy in neuronal cells
Visualize subcellular vesicles to quantitate autophagy in neuronal cells
Autophagy is an intracellular catabolic process that sequesters and degrades proteins and organelles that have either been recognized as faulty or are simply no longer needed by the cell.…
Application Note
High-content screening of neuronal toxicity using iPSC-derived human neurons
High-content screening of neuronal toxicity using iPSC-derived human neurons
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…
Scientific Poster
High-Content Assay for Morphological Characterization of Neuronal Development in 3D Matrix Using Human iPSC-Derived Neuronal Cultures
High-Content Assay for Morphological Characterization of Neuronal Development in 3D Matrix Using Human iPSC-Derived Neuronal Cultures
Development of more complex, biologically relevant, and predictive cell-based assays for compound screening is one of the main challenges in drug discovery. There is an increasing interest i…
Publications
Neurite Outgrowth in Human Induced Pluripotent Stem Cell-Derived Neurons as a High-Throughput Screen for Developmental Neurotoxicity or Neurotoxicity
Neurite Outgrowth in Human Induced Pluripotent Stem Cell-Derived Neurons as a High-Throughput Screen for Developmental Neurotoxicity or Neurotoxicity
Due to the increasing prevalence of neurological disorders and the large number of untested compounds in the environment, there is a need to develop reliable and efficient screening tools to…
Videos & Webinars

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: A New Physiologically Relevant Model for Drug Discovery Applications for High-Content Screening of Neurotoxicity

Using Electrophysiological Studies to Accelerate Mechanistic Study in Reception and Transmission

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