Protocols in Current Issue
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0 Q&A 552 Views Jan 20, 2024

The central nervous system (CNS) relies on the complex interaction of neuroglial cells to carry out vital physiological functions. To comprehensively understand the structural and functional interplay between these neuroglial cells, it is essential to establish an appropriate in vitro system that can be utilized for thorough investigation. Traditional protocols for establishing primary neuronal and mixed glial cultures from prenatal mice or neural stem cells require sacrificing pregnant mice and have the drawback of yielding only specific types of cells. Our current protocol overcomes these drawbacks by utilizing the brain from day-0 pups to isolate CNS resident neuroglial cells including astrocytes, microglia, oligodendrocytes [oligodendrocyte precursor cells (OPCs) and differentiated oligodendrocytes], and meningeal fibroblasts, as well as hippocampal neurons, avoiding sacrificing pregnant mice, which makes this procedure efficient and cost effective. Furthermore, through this protocol, we aim to provide step-by-step instructions for isolating and establishing different primary neuroglial cells and their characterization using cell-specific markers. This study presents an opportunity to isolate, culture, and establish all major CNS resident cells individually. These cells can be utilized in various cell-based and biochemical assays to comprehensively investigate the cell-specific roles and behaviors of brain resident cells in a reductionist approach.

Key features

• Efficient isolation of major neuroglial cells like meningeal fibroblasts, neurons, astrocytes, oligodendrocytes, and microglia from a single day-0 neonatal mouse pup’s brain.

• Circumvents the sacrifice of pregnant female mice.

• Acts as a bridging experimental method between secondary cell lines and in vivo systems.

• Isolated cells can be used for performing various cell-based and biochemical assays.

Graphical overview

Steps for isolation of meningeal fibroblast and neuroglial cells from day 0 pups of mice (Created using

0 Q&A 434 Views Dec 20, 2023

Satellite glial cells (SGCs) are a type of glial cell population that originates from neural crest cells. They ultimately migrate to surround the cell bodies of neurons in the ganglia of the peripheral nervous system. Under physiological conditions, SGCs perform homeostatic functions by modifying the microenvironment around nearby neurons and provide nutrients, structure, and protection. In recent years, they have gained considerable attention due to their involvement in peripheral nerve regeneration and pain. Although methods for culturing neonatal or rat SGCs have long existed, a well-characterized method for dissociating and culturing adult SGCs from mouse tissues has been lacking until recently. This has impeded further studies of their function and the testing of new therapeutics. This protocol provides a detailed description of how to obtain primary cultures of adult SGCs from mouse dorsal root ganglia in approximately two weeks with over 90% cell purity. We also demonstrate cell purity of these cultures using quantitative real-time RT-PCR and their functional integrity using calcium imaging.

Key features

• Detailed and simplified protocol to dissociate and culture primary satellite glial cells (SGCs) from adult mice.

• Cells are dissociated in approximately 2–3 h and cultured for approximately two weeks.

• These SGC cultures allow both molecular and functional studies.

Graphical overview

Dissociation and culture of mouse satellite glial cells

0 Q&A 806 Views Oct 20, 2023

During the onset of autoimmune diabetes, nerve–immune cell interactions seem to play an important role; however, there are currently no models to follow and interfere with these interactions over time in vivo or in vitro. Two-dimensional in vitro models provide insufficient information and microfluidics or organs on a chip are usually challenging to work with. We present here what we believe to be the first simple model that provides the opportunity to co-culture pancreatic islets with sympathetic nerves and immune cells. This model is based on our stamping device that can be 3D printed (STL file provided). Due to the imprint in the agarose gel, sympathetic neurons, pancreatic islets, and macrophages can be seeded in specific locations at a level that allows for confocal live-cell imaging. In this protocol, we provide the instructions to construct and perform live cell imaging experiments in our co-culture model, including: 1) design for the stamping device to make the imprint in the gel, 2) isolation of sympathetic neurons, pancreatic islets, and macrophages, 3) co-culture conditions, 4) how this can be used for live cell imaging, and 5) possibilities for wider use of the model. In summary, we developed an easy-to-use co-culture model that allows manipulation and imaging of interactions between sympathetic nerves, pancreatic islets, and macrophages. This new co-culture model is useful to study nerve– immune cell– islet interactions and will help to identify the functional relevance of neuro-immune interactions in the pancreas.

Key features

• A novel device that allows for 3D co-culture of sympathetic neurons, pancreatic islets, and immune cells

• The device allows the capture of live interactions between mouse sympatheticneurons, pancreatic islets, and immune cells in a controlled environment after six days of co-culturing.

• This protocol uses cultured sympathetic neurons isolated from the superior cervical ganglia using a previously established method (Jackson and Tourtellotte, 2014) in a 3D co-culture.

• This method requires 3D printing of our own designed gel-stamping device (STL print file provided on SciLifeLab FigShare DOI: 10.17044/scilifelab.24073062).

Graphical overview

Graphical overview of co-culture model. 1) Print the stamp with a 3D printer. 2) Isolate neurons, islets, and macrophages. 3) Use the stamp to make the imprint in the agarose gel. 4) Seed the macrophages and islets in the agarose gel on their seeding points. 5) Place the coverslip with neurons on top. 6) Incubate the culture for six days. 7) Image the co-culture. Images adapted from BioRender.

0 Q&A 364 Views Oct 5, 2023

Adult neural stem/progenitor cells (NSPCs) in two neurogenic areas of the brain, the dentate gyrus and the subventricular zone, are major players in adult neurogenesis. Addressing specific questions regarding NSPCs outside of their niche entails in vitro studies through isolation and culture of these cells. As there is heterogeneity in their morphology, proliferation, and differentiation capacity between these two neurogenic areas, NSPCs should be isolated from each area through specific procedures and media. Identifying region-specific NPSCs provides an accurate pathway for assessing the effects of extrinsic factors and drugs on these cells and investigating the mechanisms of neurogenesis in both healthy and pathologic conditions. A great number of isolation and expansion techniques for NSPCs have been reported. The growth and expansion of NSPCs obtained from the dentate gyrus of aged rats are generally difficult. There are relatively limited data and protocols about NSPCs isolation and their culture from aged rats. Our approach is an efficient and reliable strategy to isolate and expand NSPCs obtained from young adult and aged rats. NSPCs isolated by this method maintain their self-renewal and multipotency.

Key features

• NSPCs isolated from the hippocampal dentate gyrus of young adult and aged rats, based on Kempermann et al. (2014) and Aligholi et al. (2014).

• Maintenance of NSPCs isolated from the dentate gyrus of aged rats (20–24 months) in our culture condition is feasible.

• According to our protocol, maximum growth of primary neurospheres obtained from isolated NSPCs of young and aged rats took 15 and 35 days, respectively.

Graphical overview

Isolation and expansion of neural stem/progenitor cells

0 Q&A 2313 Views Jun 20, 2021

The function of neurons in afferent reception, integration, and generation of electrical activity relies on their strikingly polarized organization, characterized by distinct membrane domains. These domains have different compositions resulting from a combination of selective targeting and retention of membrane proteins. In neurons, most proteins are delivered from their site of synthesis in the soma to the axon via anterograde vesicular transport and undergo retrograde transport for redistribution and/or lysosomal degradation. A key question is whether proteins destined for the same domain are transported in separate vesicles for local assembly or whether these proteins are pre-assembled and co-transported in the same vesicles for delivery to their cognate domains. To assess the content of transport vesicles, one strategy relies on staining of sciatic nerves after ligation, which drives the accumulation of anterogradely and retrogradely transported vesicles on the proximal and distal side of the ligature, respectively. This approach may not permit confident assessment of the nature of the intracellular vesicles identified by staining, and analysis is limited to the availability of suitable antibodies. Here, we use dual color live imaging of proteins labeled with different fluorescent tags, visualizing anterograde and retrograde axonal transport of several proteins simultaneously. These proteins were expressed in rat dorsal root ganglion (DRG) neurons cultured alone or with Schwann cells under myelinating conditions to assess whether glial cells modify the patterns of axonal transport. Advantages of this protocol are the dynamic identification of transport vesicles and characterization of their content for various proteins that is not limited by available antibodies.

0 Q&A 2647 Views Mar 5, 2021

An endogenous circadian clock system enables organisms to adapt to time-of-day dependent environmental changes. In consequence, most physiological processes exhibit daily rhythms of, e.g., energy metabolism, immune function, sleep, or hormone production. Hypothalamic circadian clocks have been identified to play a particular role in coordinating many of these processes. Primary neuronal cultures are widely used as a physiologically relevant model to study molecular events within neurons. However, as circadian rhythms include dynamic molecular changes over longer timescales that vary between individual cells, longitudinal measurement methods are essential to investigate the regulation of circadian clocks of hypothalamic neurons. Here we provide a protocol for generating primary hypothalamic neuronal cultures expressing a circadian luciferase reporter. Such reporter cells can be used to longitudinally monitor cellular circadian rhythms at high temporal resolution by performing bioluminescence measurements.

0 Q&A 3980 Views Jan 5, 2021

The function of the hippocampus depends on the process of adult hippocampal neurogenesis which underpins the exceptional neural plasticity of this structure, and is also frequently affected in CNS pathologies. Thus, manipulation of this process represents an important therapeutic goal. To identify potential strategies, organotypic adult brain slices are emerging as a valuable tool. Over the recent years, this methodology has been refined and here we present a combined protocol that brings together these refinements to enable long-term culture of adult hippocampal slices. We employ a sectioning technique that retains essential afferent inputs onto the hippocampus as well as serum-free culture conditions, so allowing an extended culture period. To sustain the neurogenic potential in the slices, we utilize the gliogenesis-inhibitor Indomethacin. Using EdU retention analysis enables us to assess the effects of pharmacological intervention on neurogenesis. With these improvements, we have established an easy and reliable method to study the effects of small molecules/drugs on proliferation and neuron formation ex vivo which will facilitate future discovery driven drug screenings.

0 Q&A 3487 Views Aug 20, 2020
The deposition of misfolded, aggregated tau protein is a hallmark of several neurodegenerative diseases, collectively termed “tauopathies”. Tau pathology spreads throughout the brain along connected pathways in a prion-like manner. The process of tau pathology propagation across circuits is a focus of intense research and has been investigated in vivo in human post-mortem brain and in mouse models of the diseases, in vitro in diverse cellular systems including primary neurons, and in cell free assays using purified recombinant tau protein. Here we describe a protocol that takes advantage of a minimalistic neuronal circuit arrayed within a microfluidic device to follow the propagation of tau misfolding from a presynaptic to a postsynaptic neuron. This assay allows high-resolution imaging as well as individual manipulation of the releasing and receiving neuron, and is therefore beneficial for investigating the propagation of tau and other misfolded proteins in vitro.
1 Q&A 7117 Views Aug 5, 2020
Primary culture of mouse hippocampal neurons is a very useful in vitro model for studying neuronal development, axonal and dendritic morphology, synaptic functions, and many other neuronal features. Here we describe a step-by-step process of generating primary neurons from mouse embryonic hippocampi (E17.5/E18.5). Hippocampal neurons generated with this protocol can be plated in different tissue culture dishes according to different experimental aims and can produce a reliable source of pure and differentiated neurons in less than one week. This protocol covers all the steps necessary for the preparation, culture and characterization of the neuronal culture, including the illustration of dissection instruments, surgical procedure for embryos’ isolation, culturing conditions and assessment of culture’s purity and differentiation. Evaluation of neuronal activity was performed by analysis of calcium imaging dynamics at six days in culture.
0 Q&A 3909 Views Jan 20, 2020
Investigations into glial biology have contributed substantially in understanding the physiology and pathology of the nervous system. However, intricacies of the neuron-glial and glial-glial interactions in vivo present significant challenges while delineating the individual cell-type contributions, thus making the in vitro techniques exceedingly relevant to study glial biology. However, obtaining optimal yield along with high purity has been challenging for microglial cultures. Here we present a simple protocol to establish enriched astroglial as well as microglial cultures from the neonatal rat spinal cord. This method results in highly enriched astroglial and microglial cultures with maximal yield.

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