The authors have nothing to disclose.

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		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>5-Fluoro-2'-deoxyuridine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>F0503</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Chloride</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S9625</td>
<td>&nbsp;</td>
<tr>
<td>Vibraslicer</td>
<td>&nbsp;</td>
<td>Campden Instruments Ltd.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Potassim Chloride</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P4504</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Phosphate Dibasic</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S0876</td>
<td>&nbsp;</td>
<tr>
<td>Potassium Phosphate Monobasic</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P5379</td>
<td>&nbsp;</td>
<tr>
<td>Hepes</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H3375</td>
<td>&nbsp;</td>
<tr>
<td>D (+)-Glucose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G8270</td>
<td>&nbsp;</td>
<tr>
<td>Sucrose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S0389</td>
<td>&nbsp;</td>
<tr>
<td>Poly-D-Lysine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P7280</td>
<td>&nbsp;</td>
<tr>
<td>B27 Supplement</td>
<td>&nbsp;</td>
<td>Invitrogen (Gibco)</td>
<td>17504-044</td>
<td>&nbsp;</td>
<tr>
<td>Neurobasal Media (NBM)</td>
<td>&nbsp;</td>
<td>Invirtogen (Gibco)</td>
<td>21103-049</td>
<td>&nbsp;</td>
<tr>
<td>2-Amino-5-phosphonopentanoic acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A5282</td>
<td>&nbsp;</td>
<tr>
<td>Bovine Albumin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A7030</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin Inhibitor</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T9253</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Hydroxide, 1N solution</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>SS266-1</td>
<td>&nbsp;</td>
<tr>
<td>L-Cysteine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C7755</td>
<td>&nbsp;</td>
<tr>
<td>Bacto Agar</td>
<td>&nbsp;</td>
<td>Difco</td>
<td>0140-01</td>
<td>&nbsp;</td>
<tr>
<td>Papain</td>
<td>&nbsp;</td>
<td>Worthington </td>
<td>LS 03126</td>
<td>&nbsp;</td>
<tr>
<td>Sterile 0.2 &micro;m Syringe Filter</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>DDA02025S0</td>
<td>&nbsp;</td>
<tr>
<td>Glass Coverslips, No1, &#216;12mm</td>
<td>&nbsp;</td>
<td>Bellco Glass Inc.</td>
<td>1943-00012</td>
<td>&nbsp;</td>
<tr>
<td>Minimum Essential Media (MEM)</td>
<td>&nbsp;</td>
<td>Invitrogen (Gibco)</td>
<td>11090-081</td>
<td>with Earle's salts
without L-glutamine, needed for growing non-neuronal cultures</td>
</tr>
<tr>
<td>Penicillin/Streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen (Gibco)</td>
<td>15070-063</td>
<td>for non-neuronal culture</td>
</tr>
<tr>
<td>Fetal Bovine Serum</td>
<td>&nbsp;</td>
<td>Invitrogen (Gibco)</td>
<td>16140-071</td>
<td>needed for non-neuronal cultures</td>
</tr>
<tr>
<td>Nunclon "Triple Flask" Tissue Culture Bottle</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>12-565-25</td>
<td>Use for non-neuronal cultures. these flasks are very convenient when producing "conditioned Neurobasal Media/B27", but any other tissue culture flasks or dishes can be used instead.</td>
</tr>
<tr>
<td>Glass bottom "Imaging dishes"</td>
<td>&nbsp;</td>
<td>MatTek Corp.</td>
<td>P35G-1.5-10-C</td>
<td>Glass bottomed, &#216;35mm culture dishes ideal for Calcium or Sodium Imaging when using an inverted imaging setup. But expensive!</td>
</tr>		</tbody>
		</table>
		</div>

preparation of dissociated mouse cortical neuron cultures

וידאו זה ידריך אותך בתהליך של יצירת תרביות נוירונים בקליפת המוח של העובר מאוחר ועכבר מוקדם המוח לאחר הלידה. בתרבויות אלה יכולים לשמש עבור מגוון של יישומים, כולל immunocytochemistry, ביוכימיה, electrophysiology, הדמיה סידן, נתרן, חלבון / או RNA בבידוד. בתרבויות אלה גם לספק פלטפורמה ללימוד פיתוח העצבית של חיות טרנסגניות שנושאים מוטציה מאוחר עובריים או לאחר הלידה הגן הקטלני. ההליך הוא יחסית ישר קדימה, דורש קצת ניסיון טכניקה תרבות רקמות לא צריך לקחת יותר מ 2-3 שעות אם אתם מוכנים כראוי. הפרדה קפדנית של הקליפה קליפת המוח מתוך מערכת thalamo-קליפת המוח הסיבים יהיה להפחית את מספר הלא נוירונים תאים לא רצויים. כדי להגדיל את התשואות של תאים עצביים triturate את פיסות הרקמה קליפת המוח בעדינות אחרי שלב הדגירה האנזים. זה הכרחי שכן הוא מונע פגיעה מיותרת תאים מוקדמת מוות של תאים עצביים. מאחר תרבויות נשמרים בהעדר תאים מזין גליה, הם מציעים גם יתרון נוסף של תרבויות גדל מועשר בנוירונים.

Watch this Scientific Journal Video about Preparation of Dissociated Mouse Cortical Neuron Cultures at JoVE.com

Preparation of Dissociated Mouse Cortical Neuron Cultures

וידאו זה מציג את הליך ליצירת תרביות נוירונים מעובר מאוחר ועכבר מוקדם קליפת המוח לאחר הלידה. תרבויות אלו יכולים לשמש immunocytochemistry, ביוכימיה, electrophysiology, סידן ונתרן הדמיה לספק פלטפורמה ללימוד פיתוח העצבית של חיות טרנסגניות שנושאים מוטציה לאחר הלידה הגן הקטלני.

הכנת ניתק תרבויות עכבר קליפתיים Neuron

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures ...

preparation-of-dissociated-mouse-cortical-neuron-cultures

Universidad San Sebastian

Research

JoVE Journal

Biology

43.3K Views.  University of California, Irvine (UCI). וידאו זה מציג את הליך ליצירת תרביות נוירונים מעובר מאוחר ועכבר מוקדם קליפת המוח לאחר הלידה. תרבויות אלו יכולים לשמש immunocytochemistry, ביוכימיה, electrophysiology, סידן ונתרן הדמיה לספק פלטפורמה ללימוד פיתוח העצבית של חיות טרנסגניות שנושאים מוטציה לאחר הלידה הגן הקטלני.

Video: Preparation of Dissociated Mouse Cortical Neuron Cultures

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos and their dechorionation using bleach are demonstrated. Using a glass pipet attached to a mouth suction tube, we illustrate the removal of all cells from single embryos. The method for dispersing cells from each embyro into a small (5 l) drop of medium on an uncoated glass coverslip is demonstrated. A view through the microscope at 1 hour after plating illustrates the preferred cell density. Most of the cells that survive when grown in defined medium are neuroblasts that divide one or more times in culture before extending neuritic processes by 12-24 hours. A view through the microscope illustrates the level of neurite outgrowth and branching expected in a healthy culture at 2 days in vitro. The cultures are grown in a simple bicarbonate based defined medium, in a 5% CO2 incubator at 22-24&deg;C. Neuritic processes continue to elaborate over the first week in culture and when they make contact with neurites from neighboring cells they often form functional synaptic connections. Neurons in these cultures express voltage-gated sodium, calcium, and potassium channels and are electrically excitable. This culture system is useful for studying molecular genetic and environmental factors that regulate neuronal differentiation, excitability, and synapse formation/function.

This video demonstrates the preparation of primary neuronal cultures from midgastrula stage Drosophila embryos. Views of live cultures show cells 1 hour after plating and differentiated neurons after 2 days of growth in a bicarbonate-based defined medium. The neurons are electrically excitable and form synaptic connections.

הכנת תרבויות העצבית מ Midgastrula עוברים שלב תסיסנית

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos ...

Mouse Epidermal Neural Crest Stem Cell (EPI-NCSC) Cultures

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the physiological property to generate a wide array of differentiated cell types, including neurons, nerve supporting cells, smooth muscle cells, bone/cartilage cells and melanocytes. EPI-NCSC are easily accessible in the hairy skin and can be isolated as a highly pure population of stem cells. This video provides a detailed protocol for preparing mouse EPI-NCSC cultures from whisker follicles. The whisker pad of an adult mouse is removed, and whisker follicles dissected. The follicles are then cut longitudinally and subsequently transversely above and below the bulge region. The bulge is removed from the collagen capsule and placed in a culture plate. EPI-NCSC start to emigrate from the bulge explants 3 to 4 days later.

Mouse Epidermal Neural Crest Stem Cell EPI-NCSC Cultures

Here we show our method to isolate mouse epidermal neural crest stem cells (EPI-NCSC). Technique involves micro-dissecting whisker follicles, isolating the bulge and placeing it into tissue culture. EPI-NCSC start to emigrate from bulge explants onto the substratum within 3 - 4 days.

עכבר עוריות עצבית קרסט לתאי גזע (EPI-NCSC) תרבויות

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the ...

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation from systemic input from elsewhere in the brain. In our lab, we use these neurons to assess dopamine release kinetics using carbon fiber amperometry, as well as expression levels of dopamine related genes and proteins using quantitative PCR and immunocytochemistry. In this video, we show you how we generate these cultures from rodent neonates.
The process involves several steps, including the plating of cortical glial astrocytes, the conditioning of neuronal cell culture media by the glial substrate, the dissection of the midbrain in neonates, the digestion, extraction and plating of dopamine neurons and the addition of neurotrophic factors to ensure cell survival.
The applications suitable for such a preparation include electrophysiology, immunocytochemistry, quantitative PCR, video microscopy (i.e., of real-time vesicular fusion with the plasma membrane), cell viability assays and other toxicological screens.

Primary dissociated midbrain dopamine cell cultures allow for the study of presynaptic characteristics of dopamine neurons. They can be used to monitor real-time dopamine release kinetics and protein/mRNA levels of regulators of dopamine exocytosis. Here, we show you how to generate these cultures from rodent neonates.

ראשי ניתק דופמין במוח התיכון Cell תרבויות מן ילודים מכרסמים

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

Preparation of 2-dGuo-Treated Thymus Organ Cultures

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play a key role in the development of a functionally competent T-cell pool. However, the complexity of T-cell development in the thymus in vivo can limit analysis of individual cellular components and particular stages of development. In vitro culture systems provide a readily accessible means to study multiple complex cellular processes. Thymus organ culture systems represent a widely used approach to study intrathymic development of T-cells under defined conditions in vitro. Here we describe a system in which mouse embryonic thymus lobes can be depleted of endogenous haemopoeitic elements by prior organ culture in 2-deoxyguanosine, a compound that is selectively toxic to haemopoeitic cells. As well as providing a readily accessible source of thymic stromal cells to investigate the role of thymic microenvironments in the development and selection of T-cells, this technique also underpins further experimental approaches that include the reconstitution of alymphoid thymus lobes in vitro with defined haemopoietic elements, the transplantation of alymphoid thymuses into recipient mice, and the formation of reaggregate thymus organ cultures. (This article is based on work first reported Methods in Molecular Biology 2007, Vol. 380 pages 185-196).

This video demonstrates the dissection and removal of the fetal thymus as well the preparation of ex vivo cultures of 2-dGuo-treated thymus.

הכנת 2-dGuo שטופלו תרבויות איברים קורנית

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play ...

Preparation and Maintenance of Dorsal Root Ganglia Neurons in Compartmented Cultures

Neurons extend axonal processes that are far removed from the cell body to innervate target tissues, where target-derived growth factors are required for neuronal survival and function. Neurotrophins are specifically required to maintain the survival and differentiation of innervating sensory neurons but the question of how these target-derived neurotrophins communicate to the cell body of innervating neurons has been an area of active research for over 30 years. The most commonly accepted model of how neurotrophin signals reach the cell body proposes that signaling endosomes carry this signal retrogradely along the axon. In order to study retrograde transport, a culture system was originally devised by Robert Campenot, in which cell bodies are isolated from their axons. The technique of preparing these compartmented chambers for culturing sensory neurons recapitulates the selective stimulation of neuron terminals that occurs in vivo following release of target-derived neurotrophins. Retrograde signaling events that require long-range microtubule dependent retrograde transport have important implications for the treatment of neurodegenerative disorders.

Here we describe the technique of preparing and maintaining compartmented chambers for culturing sensory neurons of the dorsal root ganglia.

הכנה ותחזוקה של הגב הגרעינים נוירונים רוט בתרבויות מחולקים

Neurons extend axonal processes that are far removed from the cell body to innervate target tissues, where target-derived growth factors are required for ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

הכנת ראשי בתרביות תאים hematopoietic ממח העצם Murine עבור electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Preparation of Rat Brain Aggregate Cultures for Neuron and Glia Development Studies

An in vitro system that recapitulates the development and differentiation of progenitors into mature neurons and glia in the central nervous system (CNS) would provide a powerful platform for neuroscientists to investigate axo-glial interactions, properties and differentiation of multipotent progenitors, and progression of oligodendroglial lineage cells at the cellular and molecular level.  We describe here a CNS aggregate culture system from embryonic rat forebrains, which can be maintained in a serum-free medium up to 3-4 weeks and is used in our laboratory as a model to study neuron-glia interaction and CNS myelination.  This video clip will demonstrate how to isolate and grow these CNS aggregate cultures from E16 rat brain.  Furthermore, from the same brain dissection, highly enriched regular dissociated neuronal cultures can be readily obtained and used for various studies on CNS neurons or used for co-cultures with other cells. 

A protocols for an embryonic rat brain aggregate culture system is described. Multipotent progenitors in the aggregates can develop and differentiate into neurons, astrocytes and oligodendrocytes.

הכנת המוח עכברוש תרבויות מצטבר עבור Neuron ופיתוח מחקרים גליה

An in vitro system that recapitulates the development and differentiation of progenitors into mature neurons and glia in the central nervous system (CNS) ...

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons. The neurons are large (up to 1 mm in diameter) and identifiable, with distinct sizes, shapes, positions and pigmentations, and the cell bodies are externally exposed in five paired ganglia distributed throughout the body of the animal. These properties have allowed investigators to delineate the circuitry underlying specific behaviors in the animal1. The monosynaptic connection between sensory and motor neurons is a central component of the gill-withdrawal reflex in the animal, a simple defensive reflex in which the animal withdraws its gill in response to tactile stimulation of the siphon. This reflex undergoes forms of non-associative and associative learning, including sensitization, habituation and classical conditioning. Of particular benefit to the study of synaptic plasticity, the sensory-motor synapse can be reconstituted in culture, where well-characterized stimuli elicit forms of plasticity that have direct correlates in the behavior of the animal2,3. Specifically, application of serotonin produces a synaptic strengthening that, depending on the application protocol, lasts for minutes (short-term facilitation), hours (intermediate-term facilitation) or days (long-term facilitation). In contrast, application of the peptide transmitter FMRFamide produces a synaptic weakening or depression that, depending on the application protocol, can last from minutes to days (long-term depression). The large size of the neurons allows for repeated sharp electrode recording of synaptic strength over periods of days together with microinjection of expression vectors, siRNAs and other compounds to target specific signaling cascades and molecules and thereby identify the molecular and cell biological steps that underlie the changes in synaptic efficacy.
An additional advantage of the Aplysia culture system comes from the fact that the neurons demonstrate synapse-specificity in culture4,5. Thus, sensory neurons do not form synapses with themselves (autapses) or with other sensory neurons, nor do they form synapses with non-target identified motor neurons in culture. The varicosities, sites of synaptic contact between sensory and motor neurons, are large enough (2-7 microns in diameter) to allow synapse formation (as well as changes in synaptic morphology) with target motor neurons to be studied at the light microscopic level.
In this video, we demonstrate each step of preparing sensory-motor neuron cultures, including anesthetizing adult and juvenile Aplysia, dissecting their ganglia, protease digestion of the ganglia, removal of the connective tissue by microdissection, identification of both sensory and motor neurons and removal of each cell type by microdissection, plating of the motor neuron, addition of the sensory neuron and manipulation of the sensory neurite to form contact with the cultured motor neuron.

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

Primary cultures of Aplysia sensory-motor neurons provide a model preparation for studying synapse formation and synaptic plasticity in vitro. This video demonstrates the identification and microdissection of sensory and motor neurons from Aplysia ganglia as well as the methods for establishing and maintaining sensory-motor neurons in culture.

הכנת Aplysia חושי, מנוע תא תרבויות העצבית

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons.  The neurons are large (up ...

Intranuclear Microinjection of DNA into Dissociated Adult Mammalian Neurons

Primary neuronal cell cultures are valuable tools to study protein function since they represent a more biologically relevant system compared to immortalized cell lines. However, the post-mitotic nature of primary neurons prevents effective heterologous protein expression using common procedures such as electroporation or chemically-mediated transfection. Thus, other techniques must be employed in order to effectively express proteins in these non-dividing cells.
In this article, we describe the steps required to perform intranuclear injections of cDNA constructs into dissociated adult sympathetic neurons. This technique, which has been applied to different types of neurons, can successfully induce heterologous protein expression. The equipment essential for the microinjection procedure includes an inverted microscope to visualize cells, a glass injection pipet filled with cDNA solution that is connected to a N2(g) pressure delivery system, and a micromanipulator. The micromanipulator coordinates the injection motion of microinjection pipet with a brief pulse of pressurized N2 to eject cDNA solution from the pipet tip. 
This technique does not have the toxicity associated with many other transfection methods and enables multiple DNA constructs to be expressed at a consistent ratio. The low number of injected cells makes the microinjection procedure well suited for single cell studies such as electrophysiological recordings and optical imaging, but may not be ideal for biochemical assays that require a larger number of cells and higher transfection efficiencies. Although intranuclear microinjections require an investment of equipment and time, the ability to achieve high levels of heterologous protein expression in a physiologically relevant environment makes this technique a very useful tool to investigate protein function.

Direct intranuclear injection of cDNA is an effective transfection technique for post-mitotic cells. This method provides high levels of heterologous protein expression from single or multiple cDNA constructs and enables protein function to be studied in a physiologically relevant environment with a variety of single cell assays.

Microinjection Intranuclear של דנ&quot;א לתוך ניתק נוירונים היונקים למבוגרים

Primary neuronal cell cultures are valuable tools to study protein function since they represent a more biologically relevant system compared to ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

בשנת vivo ההדמיה לטיפולים במודל עכבר orthotopic של סרטן השחלות

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...