אנו מודים ברברה קארלטי אנה מארי קני הצעות שלא יסולא בפז. HYL נתמך על ידי מענק הכשרה &quot;הורמונים: לביוכימיה וביולוגיה מולקולרית&quot;, DK07328. נתמך בחלקו על ידי מענק 5R01-NIH NS16951 (רמ&quot;א).

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פרוטוקול זה מבוסס על שינויים של נהלים אשר תוארו 3,7,11 האחרונות. ישנן מספר נקודות חשובות לציין כמפורט להלן. הנוירונים גרגיר ו ובתאים לדבוק בתוך 2 שעות של ציפוי. בתאים בריאים יש מורפולוגיה עגול תחת 4 שלב הניגוד מיקרוסקופ. תוך 24 שעות לאחר ציפוי, תאים בריאים יתפשט באופן שווה סביב coverslips או גם פלסטיק יהוו תהליכים. בעת שינוי המדיום הראשון, לאחר 24 שעות, יהיה מספר תאים צפים מתים. זה נורמלי, כמו מספר תאים ימותו או יהיו בריאים במהלך תהליך הניתוק. עם זאת, אם לאחר 48 שעות התאים הם בכבדות יחד עם ורידים על התהליכים שלהם, בין אם התאים אינם בריאים, או את המצע poly-D-ליזין רעיל. כמו עם רוב מצעים, את היעילות של פולי-D-ליזין תלויה הרבה וכל הרבה חדש חייבים להיבדק. תמונות של תרבויות בריא ניתן למצוא הפניות 4,11, ו - 20. תאים בריאים יכול להתקיים בתרבות של עד שבועיים 8. נוירון אבות גרגיר להתחיל להבדיל על ציפוי 8,12,13,14. לאחר ציפוי צפיפות אופטימלית היא הוקמה ללימודי שלך, זה צריך להישמר, כי התפשטות מקודמת על ידי גורמים כגון Notch איתות 15, ותאי יתרבו יותר הצפיפויות הגבוהות. התפשטות של אבות נוירון גרגיר יכול להיות ממושך באופן משמעותי על ידי הוספת סוניק הקיפוד (ששש) ל 12,13,14 בינוני. Laminin ניתן גם להוסיף כמו מצע בנוסף poly-D-ליזין לקדם תוצאה neurite 16,17. תכונות אלה של התרבות התא גרגיר מציעים מערכת ללימוד או הביולוגיה של נוירונים גרגיר או ויסות התמיינות של תאים אבות לתוך הנוירונים 6,18,19. תאים מבודדים cerebellar מעכברים לאחר הלידה מורכבות בתחילה תערובת של נוירונים גרגיר גרגיר ו נוירון אבות בשלבים שונים של מחזור התא 8. יש גם astroglia ו interneurons נוכח בידוד / תרבות טיהור נוסף של נוירונים גרגיר גרגיר ואת אבות (95% -99%) מושגת על ידי הפרדת צבע Percoll 4. תאים מועשר גרגיר שהושג ללא שימוש הצעד ההפרדה שיפוע Percoll שימשו ללימוד הסדרת ריבוי והתמיינות של נוירון אבות גרגיר, 18,19,20. ב תימוכין לכך, אנו מוצאים כי התאים באוכלוסייה מבודדת על ידי עקיפת שלב ההפרדה Percoll שיפוע להגיב וחסונה כדי ששש ידי הנותרים מחזור התא במשך כמה ימים, וכי ללא תוספת ששש, יש ריבוי מעט מאוד בתרבויות כמצוין על ידי צביעה עם יצרנית התפשטות תאים, Ki67. עם זאת, אם התרבויות הם לשמש מעבר כמה ימים במבחנה, מומלץ כי הצעד שיפוע Percoll להיכלל להפחית זיהום של נוירונים גרגיר ידי מתרבים ללא תאים עצביים.

<table><tbody><tr><td>Cell strainer with 70&amp;mu;m mesh pore</td><td>BD Biosciences</td><td>352350</td><td></td></tr><tr><td>Spinal Needle</td><td>BD Biosciences</td><td>405182</td><td>20G x 3-1/2 inches</td></tr><tr><td>Poly-D-Lysine mol wt&amp;gt;300,000</td><td>Sigma-Aldrich</td><td>P1024</td><td>Each new batch of Poly-D-Lysine must be tested. </td></tr><tr><td>Percoll</td><td>Sigma-Aldrich</td><td>P-1644</td><td></td></tr><tr><td>Penicillin-Streptomycin (100X)</td><td>Sigma-Aldrich</td><td>P4333</td><td></td></tr><tr><td>HBSS</td><td>Invitrogen</td><td>14175-103</td><td>Must be calcium and magnesium free.</td></tr><tr><td>Neurobasal A-Medium</td><td>Invitrogen</td><td>10888-022</td><td></td></tr><tr><td>Glutamax I supplement</td><td>Invitrogen</td><td>35050-061</td><td></td></tr><tr><td>B-27 Serum Free Supplement (50x)</td><td>Invitrogen</td><td>17504-044</td><td></td></tr><tr><td>12-mm circle coverslips </td><td>Carolina Biological</td><td>63-3029</td><td>These are made in Germany by Deckgluder.</td></tr><tr><td>25-mm circle coverslips</td><td>Carolina Biological</td><td>63-3037</td><td>These are made in Germany by Deckgluder.</td></tr><tr><td>Papain Dissociation System </td><td>Worthington Biochemical</td><td>LK 003150</td><td>Kit is good for five isolations.</td></tr><tr><td>Permaset scissors</td><td>Roboz Surgical Instruments Co.</td><td>RS6782</td><td></td></tr><tr><td>Dumont #5 (Dumostar)</td><td>Roboz Surgical Instruments Co.</td><td>RS4978</td><td></td></tr><tr><td>4-well culture dish</td><td>Nalge Nunc international</td><td>176740</td><td></td></tr><tr><td>6-well culture dish</td><td>Nalge Nunc international</td><td>140685</td><td></td></tr></tbody></table>

isolation and culture of post-natal mouse cerebellar granule neuron progenitor cells and neurons 

קליפת המוח הקטן הוא מבנה תיאר היטב המספק הזדמנויות ייחודיות ללימוד המאפיינים עצב 1,2 פיתוח. סוגי נוירונים המוח הקטן (תאים גרגיר, תאים פורקינג&#39; ו interneurons מעכבות), גרגיר נוירונים הם ללא ספק רבים ביותר הן הסוג הנפוץ ביותר של הנוירונים במוח היונקים. במכרסמים, הנוירונים גרגיר cerebellar נוצרות במהלך שני הראשונים שלאחר הלידה שבועות ובתאים בשכבה החיצונית של קליפת המוח הקטן, את השכבה החיצונית גרגיר (EGL). הפרוטוקול המובא כאן מתאר טכניקות להעשיר גרגיר נוירונים תרבות ובתאים שלהם המוח הקטן שלאחר הלידה העכבר. נתאר הליכים כדי להשיג תרבויות של טוהר הגדלת 3,4, אשר ניתן להשתמש בהם כדי ללמוד את הבידול של מתרבים ובתאים לתוך הנוירונים גרגיר 5,6. לאחר ובתאים להבדיל, התרבויות גם לספק אוכלוסייה הומוגנית של נוירונים גרגיר מניפולציה אפיון ניסיוני של תופעות כגון synaptogenesis, פונקציה קולטן הגלוטמט 7, אינטראקציה עם תאים אחרים cerebellar מטוהרים 8,9 או מוות של תאים 7.

Watch this Scientific Journal Video about Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons at JoVE.com

Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons

כאן אנו מציגים שיטה לבודד cerebellar תרבות גרגר עצב ובתאים ונוירונים גרגיר cerebellar מהעכבר לאחר הלידה.

בידוד תרבות שלאחר הלידה cerebellar עכבר גרגיר Neuron ובתאים ונוירונים

The cerebellar cortex is a well described structure that provides unique opportunities for studying neuronal properties and development1,2. Of the ...

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Nanyang Technological University

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Biology

Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons 

28.5K Views.  Columbia University. כאן אנו מציגים שיטה לבודד cerebellar תרבות גרגר עצב ובתאים ונוירונים גרגיר cerebellar מהעכבר לאחר הלידה.

Video: Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons

Isolation of Distinct Cell Populations from the Developing Cerebellum by Microdissection

Microdissection is a novel technique that can isolate specific regions of a tissue and eliminate contamination from cellular sources in adjacent areas. This method was first utilized in the study of Nestin-expressing progenitors (NEPs), a newly identified population of cells in the cerebellar external germinal layer (EGL). Using microdissection in combination with fluorescent-activated cell sorting (FACS), a pure population of NEPs was collected separately from conventional granule neuron precursors in the EGL and from other contaminating Nestin-expressing cells in the cerebellum. Without microdissection, functional analyses of NEPs would not have been possible with the current methods available, such as Percoll gradient centrifugation and laser capture microdissection. This technique can also be applied for use with various tissues that contain either recognizable regions or fluorescently-labeled cells. Most importantly, a major advantage of this microdissection technique is that isolated cells are living and can be cultured for further experimentation, which is currently not possible with other described methods.

Nestin-expressing&#160;progenitors are a newly identified population of neuronal progenitors in the developing cerebellum. Using the microdissection technique presented here in combination with fluorescent-activated cell sorting, this cell population can be purified with no contamination from other cerebellar regions and can be cultured for further studies.

בידוד של אוכלוסיות תאים שונות מהמוח הקטן פיתוח על ידי Microdissection

Microdissection is a novel technique that can isolate specific regions of a tissue and eliminate contamination from cellular sources in adjacent areas. ...

Neuroscience

Spinal Cord Neurons Isolation and Culture from Neonatal Mice

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on postnatal day 1-3. A mouse litter, usually 4-10 pups born from one breeding pair, is gathered for one experiment, and spinal cords are collected individually from each mouse after euthanasia with isoflurane. The spinal column is dissected out and then the spinal cord is released from the column. The spinal cords are then minced to increase the surface area of delivery for an enzymatic protease that allows for the neurons and other cells to be released from the tissue. Trituration is then used to release the cells into solution. This solution is subsequently fractionated in a density gradient to separate the various cells in solution, allowing for neurons to be isolated. Approximately 1-2.5 x 106 neurons can be isolated from one litter group. The neurons are then seeded onto wells coated with adhesive factors that allow for proper growth and maturation. The neurons take approximately 7 days to reach maturity in the growth and culture medium and can be used thereafter for treatment and analysis.

This study presents a technique for the isolation of neurons from WT neonatal mice. It requires the careful dissection of the spinal cord from the neonatal mouse, followed by the separation of neurons from the spinal cord tissue through mechanical and enzymatic cleavage.

נוירונים חוט השדרה בידוד ותרבות מ עכברים ניאונטליים

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on ...

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their post-natal development, abundance, and accessibility, CGNs are an ideal model to study neuronal processes, including neuronal development, neuronal migration, and physiological neuronal activity stimulation. In addition, CGN cultures provide an excellent model for studying different modes of cell death including excitotoxicity and apoptosis. Within a week in culture, CGNs express N-methyl-D-aspartate (NMDA) receptors, a specific ionotropic glutamate receptor with many critical functions in neuronal health and disease. The addition of low concentrations of NMDA in conjunction with membrane depolarization to rodent primary CGN cultures has been used to model physiological neuronal activity stimulation while the addition of high concentrations of NMDA can be employed to model excitotoxic neuronal injury. Here, a method of isolation and culturing of CGNs from 6 day old pups as well as genetic manipulation of CGNs by adenoviruses and lentiviruses are described. We also present optimized protocols on how to stimulate NMDA-induced excitotoxicity, low-potassium-induced apoptosis, oxidative stress and DNA damage following transduction of these neurons.

This protocol describes a simple method for isolating and culturing primary mouse cerebral granule neurons (CGNs) from 6-7 day old pups, efficient transduction of CGNs for loss and gain of function studies, and modelling NMDA-induced neuronal excitotoxicity, low-potassium-induced cell death, DNA-damage, and oxidative stress using the same culture model.

מידול מוות עצביים והתנוונות בנוירונים גרגר אסטרוציטומה העיקרי העכבר

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...

Isolation and Cultivation of Neural Progenitors Followed by Chromatin-Immunoprecipitation of Histone 3 Lysine 79 Dimethylation Mark

Brain development is a complex process, which is controlled in a temporo-spatial manner by gradients of morphogens and different transcriptional programs. Additionally, epigenetic chromatin modifications, like histone methylation, have an important role for establishing and maintaining specific cell fates within this process. The vast majority of histone methylation occurs on the flexible histone tail, which is accessible to histone modifiers, erasers, and histone reader proteins. In contrast, H3K79 methylation is located in the globular domain of histone 3 and is implicated in different developmental functions. H3K79 methylation is evolutionarily conserved and can be found in a wide range of species from Homo sapiens to Saccharomyces cerevisiae. The modification occurs in different cell populations within organisms, including neural progenitors. The location of H3K79 methylation in the globular domain of histone 3 makes it difficult to assess. Here, we present methods to isolate and culture cortical progenitor cells (CPCs) from embryonic cortical brain tissue (E11.5-E14.5) or cerebellar granular neuron progenitors (CGNPs) from postnatal tissue (P5-P7), and to efficiently immunoprecipitate H3K79me2 for quantitative PCR (qPCR) and genome-wide sequencing.

We present an effective and reproducible method to isolate and culture neural progenitor cells from embryonic and postnatal brain tissue for chromatin immunoprecipitation (ChIP) of histone 3 lysine 79 dimethylation (H3K79me2) - a histone mark located within the globular domain of histone 3.

בידוד, טיפוח של אבות עצבית ואחריו Immunoprecipitation-כרומטין של היסטון 3 ליזין 79 Dimethylation מארק

Brain development is a complex process, which is controlled in a temporo-spatial manner by gradients of morphogens and different transcriptional programs. ...

Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model system is to provide a controlled microenvironment and maintain the high survival rate and the natural features of dissociated neuronal and nonneuronal cells as much as possible under in vitro conditions. In this article, we demonstrate a method of isolating primary neurons from the developing mouse cerebellum, placing them in an in vitro environment, establishing their growth, and monitoring their viability and differentiation for several weeks. This method is applicable to embryonic neurons dissociated from cerebellum between embryonic days 12&ndash;18.

Conducting in vitro experiments to reflect in vivo conditions as adequately as possible is not an easy task. The use of primary cell cultures is an important step toward understanding cell biology in a whole organism. The provided protocol outlines how to successfully grow and culture embryonic mouse cerebellar neurons.

התרבות הראשית של הנוירונים מבודדים מתוך עכבר ממוח עובריים

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model ...

Purification of Prominin-1+ Stem Cells from Postnatal Mouse Cerebellum

Most cerebellar neurons arise from two embryonic stem niches: a rhombic lip niche, which generates all the cerebellar excitatory glutamatergic neurons, and a ventricular zone niche, which generates the inhibitory GABAergic Purkinje cells, which are neurons that constitute the deep cerebellar nuclei and Bergman glia. Recently, a third stem cell niche has been described that arises as a secondary germinal zone from the ventricular zone niche. The cells of this niche are defined by the cell surface marker prominin-1 and are localized to the developing white matter of the postnatal cerebellum. This niche accounts for the late born molecular layer GABAergic interneurons along with postnatally generated cerebellar astrocytes. In addition to their developmental role, this niche is gaining translational importance in regards to its involvement in neurodegeneration and tumorigenesis. The biology of these cells has been difficult to decipher because of a lack of efficient techniques for their purification. Demonstrated here are efficient methods to purify, culture, and differentiate these postnatal cerebellar stem cells.

Purification of Prominin-1+ Stem Cells from Postnatal Mouse Cerebellum

Demonstrated here is an efficient and cost-effective method to purify, culture, and differentiate white matter stem cells from postnatal mouse cerebellum.

טיהור של Prominin-1+ בתאי גזע מתוך עכבר המוח פוסט לידתיים

Most cerebellar neurons arise from two embryonic stem niches: a rhombic lip niche, which generates all the cerebellar excitatory glutamatergic neurons, ...

Modeling Neuronal Injury in Mouse Primary Cerebellar Granule Neurons

Source: Laaper, M. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/55871">Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons.</a>&#160;J. Vis. Exp. (2017).
This video demonstrates the generation of a neuronal injury model in mouse primary cerebellar granule neurons. It outlines the steps involved in inducing neuronal injury using NMDA, an excitatory neurotransmitter receptor agonist, and glycine, a co-agonist. NMDA and glycine bind to specific neuronal receptors, causing their overstimulation and resulting in a massive intracellular calcium influx, which leads to neuronal cell damage and death.

מידול פגיעה עצבית בתאי עצב גרגיריים צרבלריים ראשוניים בעכבר

1. Experimental Preparation
NOTE: The following stock solutions can be prepared and maintained until use.

	Dissection Solution
	
		Dissolve 3.62 g of ...

JoVE Encyclopedia of Experiments

Neuropathology

Trauma

Modeling Reactive Oxygen Species-Induced Neuronal Death in Mouse Cerebellar Granule Neurons

Source: Laaper, M. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/55871">Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons.</a>&#160;J. Vis. Exp. (2017).
This video outlines the generation of a neuronal injury model using reactive oxygen species (ROS) in mouse primary cerebellar granule neurons. It details the process of inducing oxidative stress by adding high concentrations of hydrogen peroxide. ROS exposure causes DNA strand breaks and genomic instability. Additionally, the oxidation of cellular proteins impairs their functions, while lipid peroxidation compromises membrane integrity. This cascade leads to cytochrome c release and caspase activation, resulting in neuronal apoptosis.

מידול מוות עצבי של חמצן תגובתי בתאי עצב של גרגרי המוח הקטן בעכבר

Neurodegenerative Diseases

Isolation and Culture of Mouse Primary Cerebellar Granule Neurons

Source: Laaper, M., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/55871">Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons.</a> J. Vis. Exp. (2017)
This video demonstrates the technique for isolating and culturing primary mouse cerebellar granule neurons. Cells are treated with an antimetabolite to eliminate proliferating glial cells and produce a pure culture of cerebellar granule neurons.

בידוד ותרבית של נוירונים גרגיריים צרבלריים ראשוניים של עכבר

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Experimental Preparation
NOTE: ...

Neuronal Culture Techniques

Primary Neuronal Culture

Isolating and Culturing Murine Cerebellar Granular Neuron Progenitors

Source: Bovio, P., et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/56631">Isolation and Cultivation of Neural Progenitors Followed by Chromatin-Immunoprecipitation of Histone 3 Lysine 79 Dimethylation Mark.</a> J. Vis. Exp. (2018)
This video demonstrates the isolation and cultivation of cerebellar neural progenitor cells from a mouse pup&#39;s cerebellum. Cerebellar granule neuron progenitors are isolated and purified through differential adhesion, effectively removing astrocytes. These cultured progenitors subsequently differentiate into cerebellar granule neurons.

בידוד וטיפוח אבות נוירונים גרעיניים של המוח הקטן מורין

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1.&#160;Preparations

	Preparations ...