Name: es cell-derived neuroepithelial cell cultures
Uploaded: 2006-11-30T00:00:00
Description: Watch this Scientific Journal Video about ES Cell-derived Neuroepithelial Cell Cultures at JoVE.com

第1步<ol><li>板丝裂霉素C ggrowth抑制（10微克/毫升为2.5小时）MS5细胞明胶包30分钟（0.01％）6孔板中的α- MEM媒体的密度在70,000 /平方厘米。 </li><li>当细胞附着并已经形成了单层（过夜增长），切换至SRM。 </li><li>手动隔离ES细胞克隆胚胎干细胞的使用注射器文化与27 ½ G针。 </li><li> 1毫升的蓝色提示和低密度板MS5细胞（通常为2-3％菌落6孔板）仔细Tritrurate殖民地。 </li></ol> 注：胚胎...

<ol>
	<li>Kawasaki, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+midbrain+dopaminergic+neurons+from+ES+cells+by+stromal+cell-derived+inducing+activity.">Induction of midbrain dopaminergic neurons from ES cells by stromal cell-derived inducing activity.</a> Neuron. 28, 31-40 (2000).</li><li>Perrier, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+midbrain+dopamine+neurons+from+human+embryonic+stem+cells.">Derivation of midbrain dopamine neurons from human embryonic stem cells.</a> Proc Natl Acad Sci U S A. 101, 12543-12548 (2004).</li><li>Pruszak, J., Isacson, O., Sullivan, S., Cowan, C., Eggan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+differentiation+of+human+embryonic+stem+cells+into+dopaminergic+neurons.">Directed differentiation of human embryonic stem cells into dopaminergic neurons.</a> Human Embryonic Stem Cells: The Practical Handbook. , (2007).</li><li>Pruszak, J., Sonntag, K. C., Aung, M. H., Sanchez-Pernaute, R., Isacson, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Markers+and+methods+for+cell+sorting+of+human+embryonic+stem+cell-derived+neural+cell+populations.">Markers and methods for cell sorting of human embryonic stem cell-derived neural cell populations.</a> Stem Cells. 25, 2257-2268 (2007).</li><li>Sonntag, K. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+yield+of+neuroepithelial+precursors+and+midbrain-like+dopaminergic+neurons+from+human+embryonic+stem+cells+using+the+bone+morphogenic+protein+antagonist+noggin.">Enhanced yield of neuroepithelial precursors and midbrain-like dopaminergic neurons from human embryonic stem cells using the bone morphogenic protein antagonist noggin.</a> Stem Cells. 25, 411-418 (2007).</li><li>Kawasaki, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+dopaminergic+neurons+and+pigmented+epithelia+from+primate+ES+cells+by+stromal+cell-derived+inducing+activity.">Generation of dopaminergic neurons and pigmented epithelia from primate ES cells by stromal cell-derived inducing activity.</a> Proc Natl Acad Sci U S A. 99, 1580-1585 (2002).</li><li>Ko, J. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+embryonic+stem+cell-derived+neural+precursors+as+a+continuous,+stable,+and+on-demand+source+for+human+dopamine+neurons.">Human embryonic stem cell-derived neural precursors as a continuous, stable, and on-demand source for human dopamine neurons.</a> J Neurochem. 103, 1417-1429 (2007).</li><li>Hong, S., Kang, U. J., Isacson, O., Kim, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+precursors+derived+from+human+embryonic+stem+cells+maintain+long-term+proliferation+without+losing+the+potential+to+differentiate+into+all+three+neural+lineages,+including+dopaminergic+neurons.">Neural precursors derived from human embryonic stem cells maintain long-term proliferation without losing the potential to differentiate into all three neural lineages, including dopaminergic neurons.</a> J Neurochem. , (2007).</li><li>Zhang, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+subtype+specification+from+embryonic+stem+cells.">Neural subtype specification from embryonic stem cells.</a> Brain Pathol. 16, 132-142 (2006).</li><li>Pankratz, M. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+neural+differentiation+of+human+embryonic+stem+cells+via+an+obligated+primitive+anterior+stage.">Directed neural differentiation of human embryonic stem cells via an obligated primitive anterior stage.</a> Stem Cells. 25, 1511-1520 (2007).</li><li>Lazzari, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+derivation+of+neural+rosettes+from+cloned+bovine+blastocysts:+a+model+of+early+neurulation+events+and+neural+crest+specification+in+vitro.">Direct derivation of neural rosettes from cloned bovine blastocysts: a model of early neurulation events and neural crest specification in vitro.</a> Stem Cells. 24, 2514-2521 (2006).</li><li>Pomp, O., Brokhman, I., Ben-Dor, I., Reubinoff, B., Goldstein, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+peripheral+sensory+and+sympathetic+neurons+and+neural+crest+cells+from+human+embryonic+stem+cells.">Generation of peripheral sensory and sympathetic neurons and neural crest cells from human embryonic stem cells.</a> Stem Cells. 23, 923-930 (2005).</li></ol>

此协议表明，在生成和隔离上皮细胞从人类胚胎干细胞使用SDIA的不同步骤。这种方法的应用是多方面的，在许多协议，并已用于生产特定的神经元（如1，2，5-9）。花环被认为是像神经管细胞与前表型（2，5，10），还含有神经嵴祖细胞（11，12）。此外，他们还保留一定程度的可塑性，因为它们可以在规定的培养条件下的具体因素的图案。因此，SDIA源性神经前体细胞可以引起多种细胞类型，从中央和外周神经系?...

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		<div>
		<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>L-glutamine</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>25030</td>
<td>&nbsp;</td>
<tr>
<td>alpha-MEM</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>12571</td>
<td>&nbsp;</td>
<tr>
<td>penicillin/streptomycin</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>Knockout-DMEM</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>10829</td>
<td>&nbsp;</td>
<tr>
<td>Knockout serum replacement</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>10828</td>
<td>&nbsp;</td>
<tr>
<td>MEM non-essential amino acid solution</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>12383</td>
<td>&nbsp;</td>
<tr>
<td>DMEM/F12</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>11330</td>
<td>&nbsp;</td>
<tr>
<td>N2-A supplement</td>
<td>&nbsp;</td>
<td>Stem Cell Technologies</td>
<td>07152</td>
<td>&nbsp;</td>
<tr>
<td>mitomycin-C</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M0503</td>
<td>&nbsp;</td>
<tr>
<td>gelatine type-A</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G1890</td>
<td>&nbsp;</td>
<tr>
<td>poly-L-ornithine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P4957</td>
<td>0.01 % solution </td>
</tr>
<tr>
<td>laminin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>L-2020</td>
<td>&nbsp;</td>
<tr>
<td>fibronectin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>F2006</td>
<td>&nbsp;</td>
<tr>
<td>basic fibroblast growth factor (bFGF)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>13256</td>
<td>&nbsp;</td>
<tr>
<td>1 ml syringe with 27 1/2 G needle</td>
<td>&nbsp;</td>
<td>Becton Dickinson</td>
<td>309623</td>
<td>&nbsp;</td>
<tr>
<td>N2-A media</td>
<td>medium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>DMEM/F12 + 1% N2-A supplement </td>
</tr>
<tr>
<td>Serum replacement media (SRM)</td>
<td>medium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Knockout-DMEM + 20 % Knockout serum replacement +1% MEM non-essential amino acid solution + 2 mM L-glutamine</td>
</tr>
<tr>
<td>&#x03B1;-MEM media</td>
<td>medium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&#x03B1;-MEM + 10 % FBS + 2 mM L-glutamine + 1%penicillin/streptomycin</td>
</tr>
<tr>
<td>MS5</td>
<td>cell line</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>stromal cells </td>
</tr>
<tr>
<td>Microscope</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>6 well plates</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>for tissue culture</td>
</tr>		</tbody>
		</table>
		</div>
			<div>
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es cell-derived neuroepithelial cell cultures

胚胎干细胞有潜力分化成所有胚层，这使得他们的新疗法的发展有吸引力的工具细胞。在一般情况下，ES细胞的分化如下的概念，首先生成不成熟的祖细胞，然后就可以繁殖成成熟的细胞表型分化。这也适用于ES细胞源性神经元的神经细胞的发展，其中以下两个主要步骤：首先，推导和扩大，不成熟的神经上皮前体和第二，其分化为成熟的神经细胞。从ES细胞产生的神经前体细胞胚体（EB）的形成，从而揭示了所有胚层，包括神经外胚层细胞的分化是基于一个共同的方法。另一种更有效的方法诱导上皮细胞的发育，使用基质细胞衍生诱导活性（SDIA），可实现与颅骨骨骨髓来源的基质细胞（1）共培养的ES细胞。两个EB形成和SDIA，揭示发展的莲座状结构，这被认为是类似于神经管和/或神经嵴祖。可以分离出神经前体，扩大和进一步分化成特定的神经元和神经胶质细胞，使用定义的文化条件。在这里，我们描述的一代在与基质细胞MS5（2-5）共培养的实验和隔离等花环。

Watch this Scientific Journal Video about ES Cell-derived Neuroepithelial Cell Cultures at JoVE.com

ES Cell-derived Neuroepithelial Cell Cultures

从胚胎干细胞（ES）细胞基质细胞衍生诱导活性（SDIA）上皮前体的推导。

ES细胞源性神经上皮细胞培养

ES cells have the potential to differentiate into cells from all germ layers, which makes them an attractive tool for the development of new therapies. In ...

es-cell-derived-neuroepithelial-cell-cultures

Research

JoVE Journal

Biology

Freezing Human ES Cells

Here we demonstrate how our lab freezes HuES human embryonic stem cell lines.  A healthy, exponentially expanding culture is washed with PBS to remove residual media that could otherwise quench the Trypsin reaction.  Warmed 0.05% Trypsin-EDTA is then added to cover the cells, and the plate allowed to incubate for up to 5 mins at room temperature.  During this time cells can be observed rounding, and colonies lifting off the plate surface.  Gentle repeated pipetting will remove cells and colonies from the plate surface.  Trypsinized cells are placed in a standard conical tube containing pre-warmed hES cell media to quench remaining trypsin, and then spun.  Cells are resuspended growth media at a concentration of approximately one million cells in one mL of media, a concentration such that one frozen aliquot is sufficient to resurrect a culture on a 10cm plate.  After cells are adequately resuspended, ice cold freezing media is added at equal volume.  Cell suspensions are mixed thoroughly, aliquoted into freezing vials, and allowed to slowly freeze to -80C over 24 hours.  Frozen cells can then moved to the vapor phase of liquid nitrogen for long term storage, or remain at -80 for approximately six months.

Here we demonstrate how our lab freezes HuES human embryonic stem cell lines.

冻结人类胚胎干细胞

Here we demonstrate how our lab freezes HuES human embryonic stem cell lines.  A healthy, exponentially expanding culture is washed with PBS to remove ...

科研

生物学

Human ES cells: Starting Culture from Frozen Cells

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  approximately one (to two) million irradiated mouse embryonic fibroblast feeder cells is rinsed with HuES media to remove residual serum and cell debris, and then HuES media added and left to equilibrate in the cell culture incubator.  A frozen vial of cells from long term liquid nitrogen storage or a -80C freezer is sourced and quickly submerged in a 37C water bath for quick thawing.  Cells in freezing media are then removed from the vial and placed in a large volume of HuES media.  The large volume of HuES media facilitates removal of excess serum and DMSO, which can cause HuES human embryonic stem cells to differentiate.  Cells are gently spun out of suspension, and then re-suspended in a small volume of fresh HuES media that is then used to seed the MEF plate.  It is considered important to seed the MEF plate by gently adding the HuES cells in a drop wise fashion to evenly disperse them throughout the plate.  The newly established HuES culture plate is returned to the incubator for 48 hrs before media is replaced, then is fed every 24 hours thereafter.

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.

人类胚胎干细胞：启动文化冷冻细胞

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  ...

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

人类胚胎干细胞（ES细胞）的繁殖

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.

从Midgastrula舞台果蝇胚胎神经文化的制备

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.

小鼠表皮神经嵴干细胞（EPI的非公务员合约雇员）文化

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.

小学游离中脑多巴胺鼠类新生儿的细胞培养

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

Embryonic Stem Cell-Derived Endothelial Cells for Treatment of Hindlimb Ischemia

Peripheral arterial disease (PAD) results from narrowing of the peripheral arteries that supply oxygenated blood and nutrients to the legs and feet, This pathology causes symptoms such as intermittent claudication (pain with walking), painful ischemic ulcerations, or even limb-threatening gangrene. It is generally believed that the vascular endothelium, a monolayer of endothelial cells that invests the luminal surface of all blood and lymphatic vessels, plays a dominant role in vascular homeostasis and vascular regeneration. As a result, stem cell-based regeneration of the endothelium may be a promising approach for treating PAD.In this video, we demonstrate the transplantation of embryonic stem cell (ESC)-derived endothelial cells for treatment of unilateral hindimb ischemia as a model of PAD, followed by non-invasive tracking of cell homing and survival by bioluminescence imaging. The specific materials and procedures for cell delivery and imaging will be described. This protocol follows another publication in describing the induction of hindlimb ischemia by Niiyama et al.1

The surgical procedure for delivery of embryonic stem cell-derived endothelial cells to the ischemic hindlimb is demonstrated, with non-invasive tracking by bioluminescence imaging.

胚胎干细胞衍生的内皮细胞治疗肢体缺血

Peripheral arterial disease (PAD) results from narrowing of the peripheral arteries that supply oxygenated blood and nutrients to the legs and feet, This ...

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.

制备海兔感觉运动神经元细胞培养

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

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

原代细胞培养从果蝇原肠胚

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for investigating the development of early hematopoietic progenitor cells (HPCs). Gene expression in ESCs can be manipulated by several techniques that allow the role for individual molecules in development to be determined. One difficulty is that expression of specific genes often has different phenotypic effects dependent on their temporal expression. This problem can be circumvented by the generation of ESCs that inducibly express a gene of interest using technology such as the doxycycline-inducible transgene system. However, generation of these inducible cell lines is costly and time consuming. Described here is a method for disaggregating ESC-derived embryoid bodies (EBs) into single cell suspensions, retrovirally infecting the cell suspensions, and then reforming the EBs by hanging drop. Downstream differentiation is then evaluated by flow cytometry. Using this protocol, it was demonstrated that exogenous expression of a microRNA gene at the beginning of ESC differentiation blocks HPC generation. However, when expressed in EB derived cells after nascent mesoderm is produced, the microRNA gene enhances hematopoietic differentiation. This method is useful for investigating the role of genes after specific germ layer tissue is derived.

Manipulating temporal gene expression in differentiating embryonic stem cells (ESCs) can be achieved using inducible gene systems. However, generation of these cell lines is costly and time consuming. This protocol achieves rapid expression of a transgene in differentiating ES-derived cells and subsequent analysis of downstream hematopoietic differentiation.

小鼠胚胎的逆转录病毒感染干细胞衍生胚体细胞造血分化的分析

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for ...