The present work was supported by the German research foundation (Deutsche Forschungsgemeinschaft DFG: GRK 736, Fa 159/22-1; the research school of the Ruhr University Bochum (GSC98/1) and the priority program SSP 1172 "Glia and Synapse", Fa 159/11-1,2,3).

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The authors have nothing to disclose.

המטרה העיקרית של הפרוטוקול הנוכחי היא תרבויות עצביות ו astrocytic נפרדים לחלוטין, תוך שמירה על אותם במדיום משותף. מסיבה זו, את הטוהר של תרבויות המתקבל צריך להיות מאומת בתחילת ההליך. אנו ממליצים על שימוש של טובולין נוירון ספציפי, neurofilaments או חלבון NeuN כמו סמנים עצביים, GFAP כסמ?...

במהלך העשורים האחרונים, הפרשנות הכללית של תפקוד תאי גלייה התפתחה מן הייחוס של תומכת רק לקראת תפקיד רגולציה פעיל בדבר עצבי פונקציה 1. בגלל ההשפעה הבולטת שלהם על הומאוסטזיס המוח בבריאות ובחולי 2, האסטרוציטים הם בעלי עניין מיוחד עבור הקהילה המדעית. בשנים האחרונות, מגוון של המחקרים התמקדו נוירון, גליה אינטראקציות in vivo ו במבחנה 3. עם זאת, רוב מערכות התרבות אינו מאפשרים הניתוח הנפרד של שני סוגי התאים ושל secretomes שלהם. גישות מספר לנצל את cocultivation הישיר של נוירונים גליה להשיג הישרדות לאורך זמן ופיתוח רשת עצבית רלוונטי מבחינה פיזיולוגית 4-6. הפרוטוקול הנוכחי מגיע את אותן מטרות, תוך שמירה על סוגי תאים הן מופרדות 7 פיזית. לעומת conditioneבינוני ד גישות 8,9, המערכת שלנו מאפשרת ללמוד את בתקשורת דו-כיוונית בין הנוירונים האסטרוציטים. הביטוי של מולקולות איתות מופרשים ניתן לנטר תוך התאים לְהַבשִׁיל במדיום המשותף. הזדמנות זה רלוונטית במיוחד, כמו האסטרוציטים לשחרר גורמים מסיסים, כגון ציטוקינים, גורמי גדילה ומולקולות מטריקס 10,11, ובכך ויסות צמיחה עצבית ולתפקד 7,12. לפיכך, הוכח כי תוספת של thrombospondin לתאי הגנגליון ברשתית במבחנה גורם להיווצרות של סינפסות 13. עם זאת, אחרים עדיין גורמים לא ידועים נחוצים כדי להבהיר סינפסות 13 פונקציונלית. יתר על כן, מולקולות שפורסמו על ידי האסטרוציטים צריכות להיות מזוהות כדי להבין את הבסיס של נוירון, גליה אינטראקציות. הטיפוח של נוירונים עיקרי האסטרוציטים מעכבר והחולדה תואר בעבר 14-16. הנה אנחנולהציג כלי אלגנטים צדדי לשלב בין שני סוגי התאים בגישת coculture עקיפה. מאז שתי התרבויות מופרדים פיזית עדיין חולקים את אותו בינוני, ההשפעה של נוירונים, האסטרוציטים ומולקולות מסיסים, ניתן לנתח בנפרד, ובכך ליצור כלי רב עוצמה עבור מחקרים אינטראקציה נוירון, גליה.

<table><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>B27</td><td>Gibco (Life Technologies)</td><td>17504-044</td><td></td></tr><tr><td>Cell-culture-grade water</td><td>MilliQ</td><td></td><td></td></tr><tr><td>Cytosine-&amp;#946;-D-arabinofuranoside (AraC)</td><td>Sigma-Aldrich</td><td>C1768</td><td>CAUTION: H317, H361</td></tr><tr><td>DMEM</td><td>Gibco (Life Technologies)&amp;nbsp;</td><td>41966-029</td><td></td></tr><tr><td>DNAse</td><td>Worthington</td><td>LS002007</td><td></td></tr><tr><td>Gentamycin</td><td>Sigma-Aldrich</td><td>G1397</td><td>CAUTION: H317-334</td></tr><tr><td>Glucose</td><td>Serva</td><td>22700</td><td></td></tr><tr><td>HBSS</td><td>Gibco (Life Technologies)</td><td>14170-088</td><td></td></tr><tr><td>HEPES</td><td>Gibco (Life Technologies)</td><td>15630-056</td><td></td></tr><tr><td>Horse serum</td><td>Biochrom AG</td><td>S9135</td><td></td></tr><tr><td>L-Cysteine</td><td>Sigma-Aldrich</td><td>C-2529</td><td></td></tr><tr><td>MEM</td><td>Gibco (Life Technologies)</td><td>31095-029</td><td></td></tr><tr><td>Ovalbumin</td><td>Sigma-Aldrich</td><td>A7641</td><td>CAUTION: H334</td></tr><tr><td>Papain</td><td>Worthington</td><td>3126</td><td></td></tr><tr><td>PBS</td><td>self-made</td><td></td><td></td></tr><tr><td>Poly-D-lysine</td><td>Sigma-Aldrich</td><td>P0899</td><td></td></tr><tr><td>Poly-L-ornithine</td><td>Sigma-Aldrich</td><td>P3655</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Sigma-Aldrich</td><td>S8636</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Gibco (Life Technologies)</td><td>25300054</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>24-well-plates</td><td>Thermoscientific/Nunc</td><td>142475</td><td></td></tr><tr><td>24-wells-plate (for the&amp;nbsp; indirect co-culture)</td><td>BD Falcon</td><td>353504</td><td></td></tr><tr><td>Binocular</td><td>Leica</td><td>MZ6</td><td></td></tr><tr><td>Cell-culture inserts</td><td>BD Falcon</td><td>353095</td><td></td></tr><tr><td>Centrifuge</td><td>Heraeus</td><td>Multifuge 3S-R</td><td></td></tr><tr><td>Counting Chamber</td><td>Marienfeld</td><td>650010</td><td></td></tr><tr><td>Forceps</td><td>FST Dumont (#5)</td><td>11254-20</td><td></td></tr><tr><td>glass cover slips (12&amp;nbsp;mm)</td><td>Carl Roth (Menzel- Gl&amp;auml;ser)</td><td>P231.1</td><td></td></tr><tr><td>Incubator</td><td>Thermo Scientific</td><td>Heracell 240i</td><td></td></tr><tr><td>Micro-tube (2 mL)</td><td>Sarstedt</td><td>72,691</td><td></td></tr><tr><td>Microscope</td><td>Leica</td><td>DMIL</td><td></td></tr><tr><td>Millex Syringe-driven filter unit</td><td>Millipore</td><td>SLGV013SL</td><td></td></tr><tr><td>Orbital shaker</td><td>New Brunswick Scientific</td><td>Innova 4000</td><td></td></tr><tr><td>Parafilm</td><td>Bemis</td><td>PM-996</td><td></td></tr><tr><td>Petri dishes (10 cm)</td><td>Sarstedt</td><td>833,902</td><td></td></tr><tr><td>pipette (1 mL)</td><td>Gilson</td><td>Pipetman 1000</td><td></td></tr><tr><td>Sterile work bench</td><td>The Baker Company</td><td>Laminar Flow SterilGARD III</td><td></td></tr><tr><td>Surgical scissors</td><td>FST Dumont</td><td>14094-11</td><td></td></tr><tr><td>Syringe</td><td>Henry Schein</td><td>9003016</td><td></td></tr><tr><td>T75 flask</td><td>Sarstedt</td><td>833,911,002</td><td></td></tr><tr><td>tube (15 mL)</td><td>Sarstedt</td><td>64,554,502</td><td></td></tr><tr><td>Water bath</td><td>GFL</td><td>Water bath type 1004</td><td></td></tr></tbody></table>

the indirect neuron-astrocyte coculture assay: an in vitro set-up for the detailed investigation of neuron-glia interactions

התפתחות עצבית תקינה ותפקוד הוא התנאי ההכרחי של הפיתוח ו במוח הבוגר. עם זאת, המנגנונים להיווצרות מאוד מבוקר ותחזוקה של רשתות נוירונים מורכבות אינם מובנים לחלוטין עד כה. השאלות הפתוחות בנוגע נוירונים בבריאות ובחולי מגווני לכת להבנת ההתפתחות הבסיסית לחקירת פתולוגיות הקשורות אדם, למשל, מחלת אלצהיימר וסכיזופרניה. הניתוח המפורט ביותר של נוירונים יכול להתבצע במבחנה. עם זאת, נוירונים דורשים תאים זקוקים לתמיכה נוספת של האסטרוציטים להישרדות ארוכת הטווח. ההטרוגניות הסלולר זהו בסכסוך במטרה לנתח את הניתוח של נוירונים האסטרוציטים. אנו מציגים כאן assay תא-תרבות המאפשר cocultivation ארוכת הטווח של הנוירונים העיקריים טהור האסטרוציטים, אשר חולקים את אותה בינוני הגדרה כימית, בעת היותו פיזיתמופרד. במצב זה, התרבויות לשרוד במשך עד ארבעה שבועות assay הוא מתאים למגוון של חקירות לגבי האינטראקציה נוירון, גליה.

הניתוח של התרבויות העצביות באמצעות מערכת coculture העקיפה מגוון וניתן לבצעו בשלבים שונים של התבגרות התרבות. בשל העובדה כי התאים יכולים להישמר לתקופה של עד 4 שבועות, חקירות ארוכות טווח של התרבויות אפשריות. <p class="jove_content" fo:keep-together.within-page="1" style=";text-align:right;di...

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The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

פרוטוקול זה מתאר את coculture נוירון astrocyte עקיפות ניתוח מידור של נוירון, גליה אינטראקציות.

את assay Coculture Neuron-astrocyte העקיפה: An<em&gt; במבחנה</em&gt; הגדרה לחקירה המפורטת של נוירון, גליה אינטראקציות

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly ...

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The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

14.6K Views.  Ruhr-University.  פרוטוקול זה מתאר את coculture נוירון astrocyte עקיפות ניתוח מידור של נוירון, גליה אינטראקציות. 

Video: The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

Study Glial Cell Heterogeneity Influence on Axon Growth Using a New Coculture Method

In the central nervous system of all mammals, severed axons after injury are unable to regenerate to their original targets and functional recovery is very poor 1. The failure of axon regeneration is a combined result of several factors including the hostile glial cell environment, inhibitory myelin related molecules and decreased intrinsic neuron regenerative capacity 2. Astrocytes are the most predominant glial cell type in central nervous system and play important role in axon functions under physiology and pathology conditions 3. Contrast to the homologous oligodendrocytes, astrocytes are a heterogeneous cell population composed by different astrocyte subpopulations with diverse morphologies and gene expression 4. The functional significance of this heterogeneity, such as their influences on axon growth, is largely unknown.
To study the glial cell, especially the function of astrocyte heterogeneity in neuron behavior, we established a new method by co-culturing high purified dorsal root ganglia neurons with glial cells obtained from the rat cortex. By this technique, we were able to directly compare neuron adhesion and axon growth on different astrocytes subpopulations under the same condition.
In this report, we give the detailed protocol of this method for astrocytes isolation and culture, dorsal root ganglia neurons isolation and purification, and the co-culture of DRG neurons with astrocytes. This method could also be extended to other brain regions to study cellular or regional specific interaction between neurons and glial cells.

In this protocol, we described a new method to study the influence of glial cell heterogeneity on axon growth with an in vitro co-culture system. Rat cortical glial cells were cultured to confluence and cocultured with highly purified rat dorsal root ganglia neurons. Different glial cell influence on neurons adhesion and axon growth was compared directly in the same culture. This method provides a new way to directly study the glial cell heterogeneity influence on neuron adhesion and axon growth.

תא גלייה לימוד הטרוגניות השפעה על הצמיחה אקסון בשיטה Coculture חדש

In the central nervous system of all mammals, severed axons after injury are unable to regenerate to their original targets and functional recovery is ...

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.
Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.
A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1.
At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.
A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x106 cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.

Here we provide a protocol for culturing rat cortical neurons in the presence of a glial feeder layer. The cultured neurons establish polarity and create synapses, and can be separated from the glia for use in various applications, such as electrophysiology, calcium imaging, cell survival assays, immunocytochemistry, and RNA/DNA/protein isolation.

Bilaminar משותף התרבות של נוירונים בקליפת המוח עכברוש ראשי גליה

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

עצב Astrocyte שותף תרבות Assay לניתוח תכולה גבוהה של neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Biology

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform (MCP)-based Neuronal Axon and Glia Co-culture System

Proper neuron to glia interaction is critical to physiological function of the central nervous system (CNS). This bidirectional communication is sophisticatedly mediated by specific signaling pathways between neuron and glia1,2 . Identification and characterization of these signaling pathways is essential to the understanding of how neuron to glia interaction shapes CNS physiology. Previously, neuron and glia mixed cultures have been widely utilized for testing and characterizing signaling pathways between neuron and glia. What we have learned from these preparations and other in vivo tools, however, has suggested that mutual signaling between neuron and glia often occurred in specific compartments within neurons (i.e., axon, dendrite, or soma)3. This makes it important to develop a new culture system that allows separation of neuronal compartments and specifically examines the interaction between glia and neuronal axons/dendrites. In addition, the conventional mixed culture system is not capable of differentiating the soluble factors and direct membrane contact signals between neuron and glia. Furthermore, the large quantity of neurons and glial cells in the conventional co-culture system lacks the resolution necessary to observe the interaction between a single axon and a glial cell.
	In this study, we describe a novel axon and glia co-culture system with the use of a microfluidic culture platform (MCP). In this co-culture system, neurons and glial cells are cultured in two separate chambers that are connected through multiple central channels. In this microfluidic culture platform, only neuronal processes (especially axons) can enter the glial side through the central channels. In combination with powerful fluorescent protein labeling, this system allows direct examination of signaling pathways between axonal/dendritic and glial interactions, such as axon-mediated transcriptional regulation in glia, glia-mediated receptor trafficking in neuronal terminals, and glia-mediated axon growth. The narrow diameter of the chamber also significantly prohibits the flow of the neuron-enriched medium into the glial chamber, facilitating probing of the direct membrane-protein interaction between axons/dendrites and glial surfaces.

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform MCP-based Neuronal Axon and Glia Co-culture System

This study describes the procedures of setting up a novel neuronal axon and (astro)glia co-culture platform. In this co-culture system, manipulation of direct interaction between a single axon (and single glial cell) becomes feasible, allowing mechanistic analysis of the mutual neuron to glial signaling.

ניתוח הדמיה של תא עצב לאינטראקצית גליה בפלטפורמת תרבות Microfluidic (MCP)-מבוסס אקסון העצבי ומערכת גליה שיתוף תרבות

Proper  neuron to glia interaction is critical to physiological function of the central  nervous system (CNS). This bidirectional communication is ...

Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells

A barrier to our understanding of how various cell types and signals contribute to synaptic circuit function is the lack of relevant models for studying the human brain. One emerging technology to address this issue is the use of three dimensional (3D) neural cell cultures, termed &#39;organoids&#39; or &#39;spheroids&#39;, for long term preservation of intercellular interactions including extracellular adhesion molecules. However, these culture systems are time consuming and not systematically generated. Here, we detail a method to rapidly and consistently produce 3D cocultures of neurons and astrocytes from human pluripotent stem cells. First, pre-differentiated astrocytes and neuronal progenitors are dissociated and counted. Next, cells are combined in sphere-forming dishes with a Rho-Kinase inhibitor and at specific ratios to produce spheres of reproducible size. After several weeks of culture as floating spheres, cocultures (&#39;asteroids&#39;) are finally sectioned for immunostaining or plated upon multielectrode arrays to measure synaptic density and strength. In general, it is expected that this protocol will yield 3D neural spheres that display mature cell-type restricted markers, form functional synapses, and exhibit spontaneous synaptic network burst activity. Together, this system permits drug screening and investigations into mechanisms of disease in a more suitable model compared to monolayer cultures.

In this protocol, we aim to describe a reproducible method for combining dissociated human pluripotent stem cell derived neurons and astrocytes together into 3D sphere cocultures, maintaining these spheres in free floating conditions, and subsequently measuring synaptic circuit activity of the spheres with immunoanalysis and multielectrode array recordings.

Microcircuit סינפטית דוגמנות עם Cocultures 3D האסטרוציטים ו נוירונים מתאי גזע Pluripotent אנושי

A barrier to our understanding of how various cell types and signals contribute to synaptic circuit function is the lack of relevant models for studying ...

Developmental Biology

A Cell Culture Model for Studying the Role of Neuron-Glia Interactions in Ischemia

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent neuronal loss. Numerous evidence suggests that the interaction between glial and neuronal cells exert beneficial effects after an ischemic event. Therefore, to explore potential protective mechanisms, it is important to develop models that allow studying neuron-glia interactions in an ischemic environment. Herein we present a simple approach to isolate astrocytes and neurons from the rat embryonic cortex, and that by using specific culture media, allows the establishment of neuron- or astrocyte-enriched cultures or neuron-glia cultures with high yield and reproducibility.
To study the crosstalk between astrocytes and neurons, we propose an approach based on a co-culture system in which neurons cultured in coverslips are maintained in contact with a monolayer of astrocytes plated in multiwell plates. The two cultures are maintained apart by small paraffin spheres. This approach allows the independent manipulation and the application of specific treatments to each cell type, which represents an advantage in many studies.
To simulate what occurs during an ischemic stroke, the cultures are subjected to an oxygen and glucose deprivation protocol. This protocol represents a useful tool to study the role of neuron-glia interactions in ischemic stroke.

Here, we present a simple approach using specific culture media that allows the establishment of neuron- and astrocyte-enriched cultures, or neuron-glia cultures from the embryonic cortex, with high yield and reproducibility.

מודל תרבות תא לחקר התפקיד של אינטראקציות נוירון-גליה באיסכמיה

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent ...

 פרוטוקול ראשוני של תאי מיקרוגליה ותאי עצב למחקרי אינטראקציה של מערכת העצבים המרכזית

Generating and Co-culturing Murine Primary Microglia and Cortical Neurons

Microglia are tissue-resident macrophages of the central nervous system (CNS), performing numerous functions that support neuronal health and CNS homeostasis. They are a major population of immune cells associated with CNS disease activity, adopting&#160;reactive phenotypes that potentially contribute to neuronal injury during chronic neurodegenerative diseases such as multiple sclerosis (MS). The distinct mechanisms by which microglia regulate neuronal function and survival during health and disease remain limited due to challenges in resolving the complex in vivo interactions between microglia, neurons, and other CNS environmental factors. Thus, the in vitro approach of co-culturing microglia and neurons remains a valuable tool for studying microglia-neuronal interactions. Here, we present a protocol to generate and co-culture primary microglia and neurons from mice. Specifically, microglia were isolated after 9-10 days in vitro from a mixed glia culture established from brain homogenates derived from neonatal mice between post-natal days 0-2. Neuronal cells were isolated from brain cortices of mouse embryos between embryonic days 16-18. After 4-5 days in vitro, neuronal cells were seeded in 96-well plates, followed by the addition of microglia to form the co-culture. Careful timing is critical for this protocol as both cell types need to reach experimental maturity to establish the co-culture. Overall, this co-culture can be useful for studying microglia-neuron interactions and can provide multiple readouts, including immunofluorescence microscopy, live imaging, as well as RNA and protein assays.

מחבר זרקור: מודל תרבות משותפת במבחנה לחקר אינטראקציות מיקרוגליה-נוירונליות במצבי מחלה

This protocol describes a microglia-neuronal co-culture established from primary neuronal cells isolated from mouse embryos at embryonic days 15-16 and primary microglia generated from the brains of neonatal mice at post-natal days 1-2.

יצירה וטיפוח משותף של תאי מיקרוגליה ראשוניים של מורין ונוירונים קורטיקליים

Microglia are tissue-resident macrophages of the central nervous system (CNS), performing numerous functions that support neuronal health and CNS ...

An Indirect Neuron-Astrocyte Co-Culture Technique for Studying Neuron-Glia Interactions

Source: Gottschling, C. et al., <a href="https://app.jove.com/v/54757">The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions.</a>&#160;J. Vis. Exp.&#160;(2016)
This video illustrates a method for establishing an indirect neuron-astrocyte co-culture using a compartmentalized approach. In this setup, neurons and astrocytes are spatially separated but share a conditioned medium. Neurotrophic factors released by astrocytes promote neuron differentiation and the formation of synaptic connections, suggesting neuron-glia interactions.

טכניקה עקיפה של תרבית נוירון-אסטרוציטים לחקר אינטראקציות נוירון-גליה

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

את assay Coculture Neuron-astrocyte העקיפה: An

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תא גלייה לימוד הטרוגניות השפעה על הצמיחה אקסון בשיטה Coculture חדש

Bilaminar משותף התרבות של נוירונים בקליפת המוח עכברוש ראשי גליה

עצב Astrocyte שותף תרבות Assay לניתוח תכולה גבוהה של neurotoxicity

ניתוח הדמיה של תא עצב לאינטראקצית גליה בפלטפורמת תרבות Microfluidic (MCP)-מבוסס אקסון העצבי ומערכת גליה שיתוף תרבות

Microcircuit סינפטית דוגמנות עם Cocultures 3D האסטרוציטים ו נוירונים מתאי גזע Pluripotent אנושי

מודל תרבות תא לחקר התפקיד של אינטראקציות נוירון-גליה באיסכמיה

יצירה וטיפוח משותף של תאי מיקרוגליה ראשוניים של מורין ונוירונים קורטיקליים

יצירה וטיפוח משותף של תאי מיקרוגליה ראשוניים של מורין ונוירונים קורטיקליים

יצירה וטיפוח משותף של תאי מיקרוגליה ראשוניים של מורין ונוירונים קורטיקליים

טכניקה עקיפה של תרבית נוירון-אסטרוציטים לחקר אינטראקציות נוירון-גליה