这项工作是由美国国立卫生研究院授予NS27501 DKOD支持。授予加州大学欧文分校在霍华德休斯医学研究所教授计划通过DKOD支持这项工作提供了额外的支持。

培养前一天的准备工作： <ol><li>设为无菌解剖解决方案。 </li><li>使无菌DMEM培养液，并保持在4 ° 10毫升分装在2个星期。 </li><li>在1个月的50或100μL分装无菌DDM2补充和冻结。 </li><li>使错那县/层粘连蛋白。 </li><li>大衣盖玻片。 可选：请无菌中国建材和存储长达4个月的冻结。 </li></ol>在培养一天一，准备在层流罩酶解决方案（ES） <ol><li>把解剖解决方案（DS）在15 mL离心管5毫升。 </li><li>称取0.8毫克的L -半胱氨酸在0.6毫升的Eppendorf，加入150μL的DS。移液器向上和向下，直到晶体都不见了。 </li><li> DS /半胱氨酸的5毫升的DS加入150毫升拌匀。 </li><li>添加木瓜蛋白酶50 U（单位）。 </li><li>新增7毫升达到0.1 N娜OH拌匀。解决方案将在30分钟内清除。当被激活。 </li><li> 0.2毫米，进入无菌1.5毫升螺旋盖离心管注射器过滤器的过滤器酶液 木瓜蛋白酶：想要在5毫升的DS 50 ü。每个批次略有不同，所以必须计算多少： 37.3 mg / ml和28.3 U /毫克 37.3 x 28.3 = 1055.59单位/毫升（1000毫升） 50 U = 47.4毫升 </li></ol>二。请DDM2媒体天的培养：添加下面的补充，使DDM2 到10毫升的DMEM添加补充，只是前培养： <ol><li> 100μL转</li><li> 100μL腐胺</li><li> 100μL硒</li><li> 100μL孕酮</li><li> 50μL胰岛素</li><li>加入10μl20 - 羟基</li></ol> 注 ：所有计划中的文化（镀在一个单独的35毫米培养皿盖玻片，1.5毫秒/文化媒体每个大脑足够DDM2） 步骤III - VI族可以在完成非无菌实验室工作台上，并应在45分钟或更少完成。 三。蛹收集<ol><li>在解剖显微镜下选择10-15小瓶双方蛹。 </li><li>将蛹干35毫米培养皿的盖子。 </li></ol> 注 ：从蛹之间，我们一般使用55-78小时后puparium形成（APF）的大脑。它是从蛹年轻的脑解剖稍硬因为头部胶囊趋向崩溃，而突起生长的整体水平，是从最古老的蛹略有下降。在广- S的野生型菌株，这些阶段是公认的色素浅棕色，略微泛红的眼睛，其他地方很少颜料。 四。斩首显微镜下解剖<ol><li>将两滴无菌解剖的解决方案，在培养皿的盖子，旁边以蛹。 </li><li>用细镊子轻轻握住一个单一的蛹在左手和使用一个27号注射器针头，右手1毫升注射器在蛹前结束的角质层去除。 </li><li>稍微挤压蛹钳和头部出现，使用27号针头割断颈部。 </li><li>针或镊子尖，传输头下降解剖的解决方案。 </li><li>重复，直到你在一个下降10-15头。 </li></ol>五，去除头部的大脑解剖显微镜下<ol><li>每手举行了27号针头的1毫升注射器。 </li><li>使用这些位置在下降解剖的解决方案，所以长鼻是面对你和背水面的头部是北飞头。 </li><li>插入的长鼻留针针头部向下，使用权的针，右眼，从腹，背水面缝。 </li><li>附近地区的长鼻左侧插入正确的针，然后用留针，轻轻地抑制角质层比左眼，右眼缝推向大脑。大脑应该出现在右边的狭缝，相对完整的，经常与两个光纤LOBES仍然附着，有时色素眼组织以及。 </li><li>针转移到了一个新的无菌，35毫米培养皿无菌解剖生理盐水下降回推到大脑。 </li><li>收集各基因型10-15脑筋。 </li></ol>六。视叶和酶处理去除<ol><li>在每手按住1毫升注射器和27号针头与使用这些收集所有的大脑在一个小面积下降解剖解决方案</li><li>作为切削工具删除从每个大脑视叶，使组织文化其余是中央的大脑区域，使用注射器。 </li><li>使用20 pipetmanμL，5μL，所有的大脑的一个单一的基因型转移到Eppendorf管中含有1毫升的无菌木瓜溶液。 </li><li>大脑中的木瓜蛋白酶孵育10-15分钟在60 RPM的肩设置。 </li><li>离心3分钟3000转。 </li></ol>其余的步骤在层流罩内应该做的无菌层流罩 - 所有的地方组织暴露于空气中的步骤。 七。洗涤<ol><li>取出酶液，用无菌解剖液更换。 </li><li>离心3分钟3000转。 </li><li>用无菌解剖液洗一次，共3次。 </li><li>删除解剖生理盐水取代DDM2。 </li><li>离心机和洗共2次与DDM2。 </li><li>大脑和媒体传输到无菌的35毫米培养皿。 </li></ol>八。解剖显微镜下Trituration和电镀<ol><li>在盖玻片的中心广场5 DDM2 UL。 </li><li> 1脑转移，这有一个黄色的提示DDM2下降。 </li><li>在显微镜下，使用27表针骰子成小块（应&lt;1分钟）的大脑。 </li><li>视觉引导下，打破玻璃吸管已使大块可以轻轻吸成吸管的尖端，并驱逐到中期回拉至一个细点的提示。我们用口吸痰管，来与100μL的血细胞比容毛细管玻璃标准。 注意 ：小心不要让媒体的气泡，您将需要凭经验确定使用的最佳尺寸吸管。好的，让你的大脑在大约30秒/脑，游离于一些单个细胞，仍有一些大团块。当你看看这些在更高的功率，大约有10-20％的神经元仍然有剩下的一些过程，仍然会有一些大的团块。如果游离于太多，那么所有的细胞是圆形的，他们将有这么多的持续损害，他们将无法生存。这一步需要实践和必须迅速完成，因为有一个大的表面体积比和DDM2下降的渗透压和pH值，可以迅速改变。组织需要尽快置于二氧化碳培养箱。 </li><li>写在培养皿的盖子：“基因型，日期和时间”。 </li><li>把35毫米100毫米培养皿培养皿（湿kimwipe）和30分钟，让在CO 2培养箱解决。 </li><li>洪水1.5毫升DDM2的35毫米菜。 </li><li>保持在5％ 二氧化碳培养箱（23℃）孵化器的文化。 注意 ：如果你没有访问到冷却的CO 2培养箱，使用标准的哺乳动物组织培养的孵化器，要么把它在一个凉爽的房间和热至23 ° C，或在实验室中，临时关闭，并使用在孵化器底部的冰盘来调节周围环境温度。 </li></ol>九。喂养细胞<ol><li>在第1天（第二天），加入0.5毫升的CNBM/B27（条件培养基），使3:1 DDM2：CNBM/B27。 </li><li>第5天更换为3:1 DDM2 1毫升的媒体：CNBM/B27。 </li><li>保持在3:1 DDM2与喂养细胞：CNBM/B27，每4-5天。 注 ：文化可非神经元的空调媒体没有增长，但神经元未免显得更加脆弱，更难以在电生理学实验中使用。 </li></ol>

<ol>
	<li>Banker, G., Goslin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culturing Nerve Cells. , (1991).</li><li>Rohrbough, J., O'Dowd, D. K., Baines, R. A., Broadie, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+bases+of+behavioral+plasticity:+Establishing+and+Modifying+synaptic+circuits+in+the+Drosophila+Genetic+System.">Cellular bases of behavioral plasticity: Establishing and Modifying synaptic circuits in the Drosophila Genetic System.</a> J. Neurobiol. 54, 254-271 (2003).</li><li>O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage-gated+currents+and+firing+properties+of+embryonic+Drosophila+neurons+grown+in+a+chemically+defined+medium.">Voltage-gated currents and firing properties of embryonic Drosophila neurons grown in a chemically defined medium.</a> J. Neurobiol. 27, 113-126 (1995).</li><li>Lee, D., O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+excitatory+synaptic+transmission+mediated+by+nAChR+in+cultured+Drosophila+neurons.">Fast excitatory synaptic transmission mediated by nAChR in cultured Drosophila neurons.</a> J. Neurosci. 19, 5311-5321 (1999).</li><li>Su, H., O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+synaptic+currents+in+Drosophila+mushroom+body+Kenyon+cells+are+mediated+by+alpha-bungarotoxin-sensitive+nAChRs+and+picrotoxin-sensitive+GABA+receptors.">Fast synaptic currents in Drosophila mushroom body Kenyon cells are mediated by alpha-bungarotoxin-sensitive nAChRs and picrotoxin-sensitive GABA receptors.</a> J. Neurosci. 23, 9246-9253 (2003).</li><li>Jiang, S. A., Campusano, J. M., Su, H., O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+mushroom+body+Kenyon+cells+generate+spontaneous+calcium+transients+mediated+by+PLTX-sensitive+calcium+channels.">Drosophila mushroom body Kenyon cells generate spontaneous calcium transients mediated by PLTX-sensitive calcium channels.</a> J. Neurophysiol. 94, 491-500 (2005).</li><li>Campusano, J. M., Su, H., Jiang, S. A., Sicaeros, B., O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=nAChR-mediated+calcium+responses+and+plasticity+in+Drosophila+Kenyon+cells.">nAChR-mediated calcium responses and plasticity in Drosophila Kenyon cells.</a> Develop Neurobiol. 67, 1520-1532 (2007).</li><li>Oh, H. -. W., Campusano, J. M., Hilgenberg, L. G. W., Sun, X., Smith, M. A., O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructural+analysis+of+chemical+synapses+and+gap+junctions+between+Drosophila+brain+neurons+in+culture.">Ultrastructural analysis of chemical synapses and gap junctions between Drosophila brain neurons in culture.</a> Develop Neurobiol. , (2007).</li></ol>

收获胚胎/产后的啮齿类动物的大脑的神经细胞可以生长在原代细胞培养，他们延长突起，形成功能性突触连接。这些文化的编制方法是建立在啮齿类动物的神经元文化的研究起到了关键作用，在确定基因和环境因素参与调节突触的形成和功能的银行家和Goslin，（1991）。虽然从各种不同的物种昆虫的神经元也可以培养，从果蝇（Rohrbough等，2003）所示，形成文化功能的突触连接的唯一的昆虫神经元。神经母细胞收?...

涂布盖玻片：放置在一个60毫米的培养皿灭菌盖玻片。吸取5 UL CON /层粘连蛋白混合到每个盖玻片中心。在37℃培养箱中培养2小时。 100 UL的蒸压水冲洗3倍盖玻片。使用连接到无菌巴斯德吸管的真空。在最后一次冲洗，用血管钳拿起盖玻片和干燥双方。将盖玻片的35mm培养皿。在室温下储存长达一个月。

primary neuronal cultures from the brains of late stage drosophila pupae

在这段视频中，我们展示了从后期果蝇蛹大脑中的主要神经文化的准备。程序开始在70-78小时puparium形成后，从动物的大脑去除。木瓜蛋白酶，在无血清生长介质中的几个洗短暂的潜伏期后孤立的大脑。在盖玻片上一个媒体下降5 UL每个大脑的机械分离的过程说明。在解离附近的SOMA的有丝分裂后神经元的轴突和树突sheered但神经细胞开始再生电镀的几个小时内进程。图像显示，在2天的现场文化。神经元继续阐述在第一周在文化进程。使用GAL4线驱动组织特异性表达GFP或RFP，如荧光标记，可以识别特定的神经元的人口文化。全细胞记录已经证明，培养的神经元形成胆碱能和GABA能突触的功能，自发主动。一个简短的视频段说明使用FURA - 2作为钙指示剂监测自发性钙瞬变和一碟培养的神经元的尼古丁诱发的钙反应的培养神经元的钙动态。这些蛹脑文化是一个有用的模型系统，在其中的遗传和药理工具可以被用来确定中央突触的形成和功能的内在和外在因素影响。

Watch this Scientific Journal Video about Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae at JoVE.com

Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

这个视频演示从后期果蝇蛹大脑中的主要神经文化的准备。生活文化的视图显示突起生长的钙水平使用FURA - 2和成像。

从晚期果蝇蛹的大脑原发性神经文化

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the ...

primary-neuronal-cultures-from-brains-late-stage-drosophila

Research

JoVE Journal

Biology

15.0K 次观看.  University of California, Irvine (UCI). 这个视频演示从后期果蝇蛹大脑中的主要神经文化的准备。生活文化的视图显示突起生长的钙水平使用FURA - 2和成像。

视频: Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

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 ...

科研

生物学

Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information into directional movement towards the appropriate target cell. To fully understand how guidance information is transmitted from the cell surface to the underlying dynamic cytoskeletal networks, one needs a model system suitable for live cell imaging of protein dynamics at high temporal and spatial resolution. Typical vertebrate growth cones are too small to quantitatively analyze F-actin and microtubule dynamics. Neurons from the sea hare Aplysia californica are 5-10 times larger than vertebrate neurons, can easily be kept at room temperature and are very robust cells for micromanipulation and biophysical measurements. Their growth cones have very defined cytoplasmic regions and a well-described cytoskeletal system. The neuronal cell bodies can be microinjected with a variety of probes for studying growth cone motility and guidance. In the present protocol we demonstrate a procedure for dissection of the abdominal ganglion, culture of bag cell neurons and setting up an imaging chamber for live cell imaging of growth cones.

Aplysia californica neurons develop large growth cones in culture that are excellent for high-resolution imaging of growth cone motility and guidance. Here, we present a protocol for dissection and plating of Aplysia bag cell neurons as well as for setting up a chamber for live cell imaging.

从海兔的神经细胞培养的高分辨率成像生长锥

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information ...

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 ...

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.

主电穿孔的小鼠骨髓造血细胞培养制备

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 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 ...

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three preparations of the brain and visual system that cover the range from the developing eye disc-brain complex in the developing pupae to individual eye and brain dissection from adult flies. All protocols are optimized for the live culture of the preparations. However, we also present the conditions for fixed tissue immunohistochemistry where applicable. Finally, we show live imaging conditions for these preparations using conventional and resonant 4D confocal live imaging in a perfusion chamber. Together, these protocols provide a basis for live imaging on different time scales ranging from functional intracellular assays on the scale of minutes to developmental or degenerative processes on the scale of many hours.

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

This protocol describes three Drosophila preparations: 1) adult brain dissection, 2) adult retina dissection and 3) developing eye disc- brain complexes dissection. Emphasis is laid on special preparation techniques and conditions for live imaging, although all preparations can be used for fixed tissue immunohistochemistry.

发展和成人的制备果蝇大脑和视网膜实时成像

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three ...

Establishing Primary Adult Fibroblast Cultures From Rodents

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized. Primary cells are preferred in studies of cell cycle control, apoptosis, and DNA repair, as cancer cells carry mutations in genes involved in these processes. Primary cells cannot be cultured indefinitely due to the onset of replicative senescence or aneuploidization. Hence, new cultures need to be established regularly. The procedure for isolation of rodent embryonic fibroblasts is well established, but isolating adult fibroblast cultures often presents a challenge. Adult rodent fibroblasts isolated from mouse models of human disease may be a preferred control when comparing them to fibroblasts from human patients. Furthermore, adult fibroblasts are the only available material when working with wild rodents where pregnant females cannot be easily obtained. Here we provide a protocol for isolation and culture of adult fibroblasts from rodent skin and lungs. We used this procedure successfully to isolate fibroblasts from over twenty rodent species from laboratory mice and rats to wild rodents such as beaver, porcupine, and squirrel.

This article describes a protocol for isolation and maintenance of primary fibroblast cultures from skin and lung tissue of wild rodents.

建立从小学成人成纤维细胞培养鼠害

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized.  Primary cells are preferred ...

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 ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&amp;#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

天真成人干细胞的分离从初级人类成纤维细胞培养

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

The Utility of Stage-specific Mid-to-late Drosophila Follicle Isolation

Drosophila oogenesis or follicle development has been widely used to advance the understanding of complex developmental and cell biologic processes. This methods paper describes how to isolate mid-to-late stage follicles (Stage 10B-14) and utilize them to provide new insights into the molecular and morphologic events occurring during tight windows of developmental time. Isolated follicles can be used for a variety of experimental techniques, including in vitro development assays, live imaging, mRNA expression analysis and western blot analysis of proteins. Follicles at Stage 10B (S10B) or later will complete development in culture; this allows one to combine genetic or pharmacologic perturbations with in vitro development to define the effects of such manipulations on the processes occurring during specific periods of development. Additionally, because these follicles develop in culture, they are ideally suited for live imaging studies, which often reveal new mechanisms that mediate morphological events. Isolated follicles can also be used for molecular analyses. For example, changes in gene expression that result from genetic perturbations can be defined for specific developmental windows. Additionally, protein level, stability, and/or posttranslational modification state during a particular stage of follicle development can be examined through western blot analyses. Thus, stage-specific isolation of Drosophila follicles provides a rich source of information into widely conserved processes of development and morphogenesis.

The Utility of Stage-specific Mid-to-late Drosophila Follicle Isolation

Stage-specific isolation of mid-to-late Drosophila follicles is useful for a variety of purposes. Such follicles develop in culture, which allows for genetic and/or pharmacologic manipulations to be coupled with in vitro development assays and live imaging. Additionally, follicles can be used for molecular studies, such as isolating mRNA and protein.

的效用舞台专用中旬至下旬<em&gt;果蝇</em&gt;卵泡隔离

Drosophila oogenesis or follicle development has been widely used to advance the understanding of complex developmental and cell biologic processes. This ...