Neurite growth analysis software tools laser scanning microscopy image analysis in vitro assays to measure neuronal growth are a fundamental tool used by many neurobiologists studying neuronal development and regeneration. This biological phenomena is referred to as neurite outgrowth and is regulated by complex intracellular signaling events. Neurite outgrowth is a process wherein developing neurons produce new projections as they grow in response to guidance cues. The total length of neurite growth in ngf or igf did not differ significantly from each other, although both were different from the controls p neurite outgrowth is a process wherein developing neurons produce new projections as they grow in response to guidance cues.
To improve the outcomes of individuals with neurodegenerative diseases and injury, it is necessary to understand and develop optimal extracellular signals that can induce neuronal regenerative. Application note 14000535 aa in cell developer toolbox. Testing procedure for simple neurite tracer imagej. Although, in general many constituents of the machinery involved in this multistage process are common for neurons in distinct brain areas, there are notable differences between. Hopach clustering and genmapp annotation of genes changed in both drg and scg during neurite outgrowth in vitro. The collagen gel system provides neurite outgrowth conditions that mostly close mimics conditions in vivo harris et al. Neurite analysis incucyte applications essen bioscience. Images from retinoic acidinduced neurite outgrowth were acquired with the in cell analyzer 3000 and analyzed by both the in cell analyzer 3000 neurite outgrowth image analysis module and the in cell developer toolbox. A facile method for simultaneously measuring neuronal cell.
We first investigated whether enlargeosomes are expressed and exocytized by wtpc12 cells during treatment with ngf, basic fgf, or egf 14. Related content neurite outgrowth at the interface of 2d and 3d growth environments celinda m kofron, vivian j fong and diane hoffmankim. Livecell analysis system and incucyte neurotrack software module cat. Differentiating human luhmes cells were used as potential model for. Assessment of chemical effects on neurite outgrowth in. Validation of neurotrack assay for mediumthroughput testing in 96. Organspecific in vitro toxicity assays are often highly sensitive, but they lack specificity. With the incucyte livecell analysis system and incucyte neurotrack software module cat. Dynamic neurite outgrowth during development results in the formation of a complex neuronal architecture that results in the. Understanding the signaling mechanisms driving neurite outgrowth provides valuable insight into neurotoxic responses, compound screening, and for interpreting factors influencing neural regeneration. Using the oligomannosemimicking peptide in crosslinking experiments, synapsin i was. Incucyte neurite analysis assays enable automated, continuous analysis of neurite outgrowth using your choice of neurons e.
Jun 29, 2018 the results of neurite outgrowth in the present study suggest that this 3d primary neuronal culture is a reliable in vitro model of the disease model of neurite outgrowth deficit derived from the. Hcs neurite outgrowth assay thermo fisher scientific us. The enlargeosome marker da appeared in the cells, and at their surface, after 6 h of treatment, stronger with ngf than with the other factors fig. The plugin has builtin tools for analysis and hardware accelerated 3d visualization of the results. When combined with the speed and ease of thermo scientific hca platforms, you can get your results fast. Apr 26, 2006 neurite outgrowth is mediated by the exocytosis of intracellular vesicles at the tips of elongating neuronal processes.
Uridine from pleurotus giganteus and its neurite outgrowth. Simplify characterization of neurons using automated cell imaging and preconfigured neurite outgrowth analysis protocol. Neurons create connections via extensions of their cellular body called processes. Decreasing the media serum concentration from 8% to 2% promotes neurite outgrowth. Assay conditions in which staurosporine is added 1 day after plating, followed by 3 days of incubation, resulted in zfactors z for neurite parameters that ranged from 0.
An edible mushroom, pleurotus giganteus was found to stimulate neurite outgrowth in vitro but the chemical constituents and the underlying mechanism is yet to be elucidated. The neurite outgrowth kit reagents, in combination with the arrayscan hcs reader and the extended neurite outgrowth bioapplication software enable automated plate handling, focusing, cell image acquisition, analysis, and neurite outgrowth quantification. Automated analysis of neurite outgrowth in mouse retinal. Neurodegenerative diseases are linked to neuronal cell death and impairment of neurite outgrowth. Neurite outgrowth in vitro neurotoxicity assays cyprotex. Imagexpress micro 4, high content screening widefield. The enlargeosome marker da appeared in the cells, and at their surface, after 6 h of treatment, stronger with ngf than with the other.
Understanding the signaling mechanisms driving neurite outgrowth provides valuable insight into neurotoxic responses, compound screening, and for interpreting factors influencing. Following image capture, all image analysis was accomplished using the cellreporterxpress automated image acquisition and analysis software, which contained image processing application modules for neurite outgrowth and viability assessment. Neurite outgrowth and cell health analysis protocol. Total neurite growth represents the sum of all traced segments. Highthroughput screen for compounds that modulate neurite. After treatment with test articles and control compounds, neurons are maintained in a humidified environment at 37c with 5% co 2 for 72 hrs optimal.
Nerve growth factors, or neurotrophins, are one family of such stimuli that regulate neurite growth. A number of commercial highcontent cellular imaging systems include software that can characterize neuronal projections i. Thermo scientific hcs studio cell analysis software has over 30 builtin applications for neurite detection, neurite outgrowth, synaptogenesis assays, viability. Neuronal cell culture, chemicals, neurite outgrowth, neurite tracing. Contactassociated neurite outgrowth and branching of. Neurite outgrowth is a key process in the development of functional neuronal circuits and the regeneration of the nervous system following injury. Data can be imported and exported in swc files for interaction with other software, or details of the. In the past, students have proposed students ttests and oneway. In vitro neurite outgrowth assay for assessing druginduced neurotoxicity and. Jan 19, 2016 time lapse, live cell imaging of neurite outgrowth, performed in the laboratories of cell applications, inc. Proceed to section 3, software preparation and image analysis, if the. This biological phenomenon is referred to as neurite outgrowth. Electrical stimulation with a conductive polymer promotes.
The video used a nanoentek juli stage analyzer to directly acquire cell. The synaptotagmin syt proteins have been widely studied for their role in regulating fusion of intracellular vesicles with the plasma membrane. Measuring changes in neurite outgrowth is one of the most widely performed phenotypic assays since the ability of neuronal cells to project membrane extensions from their cell bodies is closely linked to their function and cell health 1 harrill ja, robinette bl, freudenrich t, mundy wr. Please use one of the following formats to cite this article in your essay, paper or report. Analysis of gene expression during neurite outgrowth and. Neurite definition of neurite by medical dictionary. In purely excitatory networks, for example, when the level of activity is too high, the neuron will withdraw neurites and, consequently. This kit includes fluorescent antibodies, buffers, and a complete protocol for preparation of imagingquality cells in microplates. Although, in general many constituents of the machinery involved in this multistage process are common for neurons in distinct brain areas, there are notable differences between specific neuronal subtypes. A role for synaptotagmin viiregulated exocytosis of. Induction of neurite outgrowth in pc12 cells treated with. Using this technology, neurite outgrowth can be quantified in each well derived from hundreds of cell measurements in a 96well microplate in approximately 30 min. Analysis tutorial for cellpathfinder neurite outgrowth yokogawa. Perform detailed morphometric analysis of neurons, such as quantifying.
For the impaired axonal outgrowth in hsp, the measurement of neurite outgrowth parameters can be automatically quantified using existing software wang et al. Testing procedure for simple neurite tracer this is a set of instructions to follow to test as much of the code as possible in simple neurite tracer. Nov 25, 2014 enlargeosome exocytosis in the ngfinduced neurite outgrowth. The purpose of the current study is to follow the early events of interneuron neurite outgrowth and branching in a threedimensional collagen gel environment. We chose a potentially stimulatory cell stretch condition 10%, 0. At the end of the treatment period, cells are fixed, permeabilized and stained for evaluation of neurite outgrowth and cell health. The molecular probes neurite outgrowth staining kit allows for quick and simple measurement of neurite outgrowth and cell viability in the same sample. Screening for chemical effects on cell proliferation, neurite outgrowth and viability can be achieved in a highthroughput format protocols were developed for screening and prioritization of chemicals for further testing that may reduce the demands associated with toxicity testing in vivo.
The neurite outgrowth instigated by bucladesine in cell cultures has been shown to be enhanced by nardosinone. Enlargeosome exocytosis in the ngfinduced neurite outgrowth. Neurite outgrowth is a key process during neuronal migration and differentiation. The impact of laminin on 3d neurite extension in collagen gels. A tool for creating and analyzing realistic, meaningful, and quantifiable neuron reconstructions from microscope images.
Neurons create connections via extensions of their cellular body called dendrites or processes. Complex intracellular signaling is involved in the initiation of neurite protrusion and subsequent elongation. Neurons are plated on laminincoated 384well plates 1 hour prior to treatment. Ferulic acid protects against lead acetateinduced inhibition of neurite outgrowth by upregulating ho1 in pc12 cells. Differentiating human luhmes cells were used as potential model for developmental neurotoxicity testing. A the cluster displays the averaged affymetrix microarray data for 5 time points of drg outgrowth 2, 5, 12, 24, and 40 hours and 6 time points of scg outgrowth same as drg plus 65 hour endpoint, for the 712 probe sets found to change more than 1.
Synapsin i is an oligomannosecarrying glycoprotein, acts. Maximal neurite outgrowth is typically observed at fbs concentrations between 0. For the impaired axonal outgrowth in hsp, the measurement of neurite outgrowth parameters can be automatically quantified using existing software wang et. Signaling involved in neurite outgrowth of postnatally. It does not give complete coverage, but following this before each new release should stop the more obvious problems from getting through. We evaluated here examples of assay features that can affect test specificity, and some general procedures are suggested on how positive hits in complex biological assays may be defined. Dynamic neurite outgrowth during development results in the formation of a complex neuronal architecture that results in the establishment of the. The collagen gel system provides neurite outgrowth conditions that mostly close mimics conditions in. The results of neurite outgrowth in the present study suggest that this 3d primary neuronal culture is a reliable in vitro model of the disease model of neurite outgrowth deficit derived from the. Values obtained by neuritetracer correlate strongly with those obtained by semimanual tracing with neuronj and by using a sophisticated analysis package, metaxpress. The goal of the present study was to evaluate the utility of an automated high content screening system, the cellomics arrayscan, to detect chemical effects on neurite outgrowth. Thermo scientific hcs studio cell analysis software has over 30 builtin applications for neurite detection, neurite outgrowth, synaptogenesis assays, viability, and more.
Feb 05, 2020 together with in vivoneurite outgrowth data in figure 5c and d, these data support the conclusion that trc and pavarotti tailor neurite outgrowth together, by controlling microtubule sliding. Inducing neurite outgrowth by mechanical cell stretch. Characterization of neurite outgrowth, maturation, and the disruption of neurite. The faster systems, capable of running at primary screening speeds, tend to measure only the simplest feature describing these neurite out. Using imagej to assess neurite outgrowth in mammalian cell. In addition, neuritetracer is able to detect the neurite outgrowth promoting capacity of the rho kinase inhibitor y27632. Gene expression observed in these models likely includes both developmental gene expression patterns and regenerative responses to axotomy, which occurs as the result of tissue dissection.
By performing a phage display screening with oligomannosespecific antibodies, we identified an oligomannosemimicking peptide that was functionally active in modulating neurite outgrowth and neuronastrocyte adhesion. Together with in vivoneurite outgrowth data in figure 5c and d, these data support the conclusion that trc and pavarotti tailor neurite outgrowth together, by controlling microtubule sliding. Validation of neurotrack assay for mediumthroughput. In this tutorial, we will observe the change in number and length of neurites due to nerve growth factor ngf stimulation in pc12 cells. The faster systems, capable of running at primary screening speeds, tend to measure only the simplest. Automated image analysis is becoming a central tool for quantifying the effect of candidate drugs on cells. The lysosomal vesicleassociated soluble n ethylmaleimidesensitive factor attachment protein receptor tetanus neurotoxin insensitive vesicleassociated membrane protein tivampvamp7 was previously implicated in membrane fusion events mediating neurite outgrowth, but the.
Data can be imported and exported in swc files for interaction with other software. Use of high content image analyses to detect chemicalmediated effects on. Screening for chemical effects on neuronal proliferation. May 18, 2011 oligomannosidic glycans play important roles in nervous system development and function. May 14, 20 organspecific in vitro toxicity assays are often highly sensitive, but they lack specificity. Thermo scientific neurite outgrowth reagent kit from. We have undertaken a genomewide study of transcriptional activity in embryonic superior cervical ganglia scg and dorsal root ganglia drg during a time course of neurite outgrowth in vitro. Cyprotexs neurite outgrowth assay uses high content screening hcs technology to monitor neurite outgrowth. Neurite outgrowth experiments are therefore often analyzed by laborintensive manual or semimanual tracing meijering et al. Synaptotagmin 17 controls neurite outgrowth and synaptic.
Published 24 january 2007 2007 iop publishing ltd journal of neural engineering, volume 4, number 2. The lysosomal vesicleassociated soluble n ethylmaleimidesensitive factor attachment protein receptor tetanus neurotoxin insensitive vesicleassociated membrane protein tivampvamp7 was previously implicated in membrane fusion events. The neurite outgrowth kit labels the neuronal form of tubulin to detect neurons and neurites, to enable assessment of neurite outgrowth. Neurite outgrowth is mediated by the exocytosis of intracellular vesicles at the tips of elongating neuronal processes. Jennie b leach 1,3, xin q brown 1, jeffrey g jacot 1,4, paul a dimilla 1,2 and joyce y wong 1. The impact of laminin on 3d neurite extension in collagen gels to cite this article. Imagexpress pico system with cellreporterxpress software thumbnail view of neurite outgrowth. A the cluster displays the averaged affymetrix microarray data for 5 time points of drg outgrowth 2, 5, 12, 24, and 40 hours and 6 time points of scg outgrowth same as drg plus 65 hour endpoint, for the 712 probe sets found to change more. Model studies have shown that this activitydependent neurite outgrowth will contribute to the homeostasis of neural activity, both in purely excitatory and in mixed networks of excitatory and inhibitory cells. Snt is designed to allow easy semiautomatic tracing of neurons or other tubelike structures e.
As an example of image processing, figure 1 shows zoomed in representative images from neurite images. Evaluation of a human neurite growth assay as specific. After treatment with test articles and control compounds, neurons are maintained. Oct 03, 2016 please use one of the following formats to cite this article in your essay, paper or report. Neurite outgrowth and branching of pc12 cells on very soft substrates sharply decreases below a threshold of substrate rigidity. Time lapse, live cell imaging of neurite outgrowth, performed in the laboratories of cell applications, inc. Pdf using imagej to assess neurite outgrowth in mammalian. Neurite outgrowth, neurotoxicity molecular devices. Nerve growth factors, or neurotrophins, are one family of such stimuli that regulate neurite growth 1. Neurite outgrowth and branching of pc12 cells on very soft.
Sep 01, 2005 automated image analysis is becoming a central tool for quantifying the effect of candidate drugs on cells. The in cell analyzer 3000 software analyzes neurite outgrowth on the basis of population i. Neuronal plasticity can be defined as the ability of the nervous system to change structure, function and organization of neurons in response to new experiences, emerging from changes of developmental and environmental situations, as well as other factors e. Gene expression observed in these models likely includes both developmental gene expression patterns and regenerative responses to axotomy, which occurs as the result. Oligomannosidic glycans play important roles in nervous system development and function. Neurite growth software tools laser scanning microscopy. As a consequence of the limited availability of automated software for neurite outgrowth analysis of tissue explants, most researchers resort to manual analysis methods, such as the maximum neurite length method,14 and the neurite outgrowth area, 22,23 both mentioned above, or to semiautomatic methods, typical for neurite area detection. To develop an accessible tool for automated tracing, we have written neuritetracer, a plugin for the multiplatform free imageprocessing program, imagej abramoff et al.
1307 1461 445 1407 465 403 1034 1380 344 1182 540 1100 1618 1347 1518 599 299 435 185 644 751 1560 1444 840 43 1295 406 1014 735 609 1648 197 385 1104 621 1385 1115 857 949 627 210 1448 352 927 622 496 765 1395 1375 851