巴顿得到了达蒙Runyon癌症研究基金会奖学金DRG - 1716 - 02。锦州是支持由美国国立卫生研究院/ NIGMS奖学金F32 GM082174 - 02。 NP是霍华德休斯医学研究所研究员。

1。准备蝇笼<ol><li>生长在方便的容器，如32盎司昆虫杯果蝇（野生型菌株，如俄勒冈- R或任何基因型）。 </li><li>新eclosed苍蝇大的胚胎回收笼，或飞人口笼。 </li><li>一个房间移动到笼子里的温度〜25 ° C，湿度〜65％在12小时轻和黑暗的12小时周期，以方便收集同步胚胎。 </li><li>维持在笼子里的苍蝇喂养它们杀死酵母膏挑染糖蜜板。糖蜜板（死亡人数酵母膏）为2-3天更换一次D. </li></ol> 2。收集同步胚胎<ol><li>在原细胞制备的一天，饲料苍蝇杀害酵母WTH新鲜涂层钢板为1-2 h前奠定期间粘贴。 </li><li>就在12小时黑暗周期，更换新鲜糖蜜与少量的酵母膏挑染板前打下板。丢弃前打下的集合，它包含异步年纪较大的胚胎。 </li><li>收集胚胎期为1-2小时。 </li><li> 5-6小时取出板含有胚胎和存储相对同步的人口在25 ° C胚胎将在先进的原肠胚阶段，在这个时候。 </li></ol> 3。胚胎收集和洗涤<ol><li>松开的胚胎，用湿画笔板块，并通过一个筛子叠不温不火的自来水彻底冲洗，使胚胎在底部收集最好的目筛。冲洗几分钟，以消除尽可能多的酵母和飞行碎片。 </li><li>提示的筛子，使胚胎在一侧积累，并轻轻从筛洗成dechorionation筐。 </li><li>移动到组织文化引擎盖区，而让胚胎干出。 </li></ol> 4。同质化的胚胎和细胞电镀<ol><li>他们沉浸在50％（体积/体积）5-10分钟漂白消毒和dechorionate胚胎。篮子的墙上用清水冲洗干净，用70％乙醇（体积/体积）的胚胎。另外，用蒸馏水洗净漂白乙醇治疗前关闭胚胎。从这一点起胚胎应在无菌条件下和在组织培养罩处理。 </li><li>冲洗胚胎彻底灭菌蒸馏水中删除的漂白剂和/或乙醇。 </li><li>在dechorionation步骤，填写一个Dounce匀浆，室温供应M3的介质的基础上，胚胎数量适当的音量。之间的胚胎数量和比例的媒体不应该超过0.5毫升胚胎：40毫升介质（或约100-200胚胎/毫升）。 </li><li> M3的媒体的数量足以淹没胚胎放置在灭菌烧杯冲洗胚胎。让胚胎浸泡2-5分钟。 </li><li>拆开dechorionation篮子和印迹Nitex网状消毒纸巾擦干。 </li><li>胚胎转移到均质，灭菌画笔。 </li><li>均质轻轻短行程而深远的试管底部，直到所有的胚胎暂停使用一个松散的杵。然后轻轻地，但坚定地，同质化八分满招。杵为向上和向下移动，不要扭曲。 </li><li>通过浇注或移液匀浆转移到无菌的50毫升锥形离心管，并帽管。 </li><li>离心10分钟40G，室温组织碎片，大颗粒细胞团块和卵黄膜。转移上清到一个干净的管含有细胞和蛋黄和重复离心5分钟步。 </li><li>小心地浇或360克在10分钟到一个干净的管和自旋吹打，室温颗粒细胞转移上清。上清液。悬浮在10毫升培养液中的细胞沉淀（RNAi实验的无血清培养基）。 </li><li>使用血球估计细胞密度的活菌计数。同时，重复步骤4.10颗粒细胞。 </li><li>悬浮细胞至所需浓度。要启动，请尝试范围是1〜5 × 10 6细胞/ ml和板在1.7至2.5 × 10 5细胞/厘米2。 </li></ol> 5。在384孔板细胞生长的主要细胞的RNAi <ol><li>免除到每口井在〜250纳克在384孔板，每孔终浓度为5μLdsRNAs。 </li><li>使用多道移液器，免除每口井的主要细胞在无血清M3中型384板，包含在每口井的不同的双链RNA的10毫升（1〜4x106细胞/ ml）。此外，细胞可以培养8以及玻璃共焦成像玻片。 </li><li>离心机板300G，室温1分钟。培养细胞无血清培养基在25 ° C为22小时或18 ° 1-2彗星D. </li><li>含血清培养基中加入30毫升，每口井。 </李&gt; <li>离心机板300G，室温1分钟。 </li><li>板放置在潮湿室和文化的主要细胞为额外的5〜7天在25 ° C。 </li><li>图片的细胞免疫荧光染色后的活。 </li></ol> 6。代表性的成果： 在最佳条件下，在文化的细胞看起来很健康的，圆的形态。文化将有小细胞碎片，并且50〜80％融合后初步电镀。这些细胞经过长时间培养后的形态学变化。它通常需要5-8天左右的文化细胞得到充分分化在25 ° C。优质的小学文化通常是判断细胞的利益如何在文化区别。例如，小学的肌肉细胞往往有分支状的形态，并成为与条纹状的肌系列组成的肌原纤维的多核肌管。完全分化的肌管通常会自发地在文化移动。相反，差异化的初级神经元形成的细胞体的集群，其轴突电缆连接与其他集群。 <img src="/files/ftp_upload/2215/2215fig1.jpg" alt="图1" /> 图1。主细胞进行形态学变化后，在文化镀 （A）原代细胞的明场像，后在384孔板以及镀。多数细胞是全面和完整，并很好地分离。 （B）原代细胞培养20小时后镀亮场图像。原代细胞已经形成集群（大箭头）的一个分支状的形态（长箭头），可以在此阶段的肌肉。 （三）一个小学文化的形象相衬，电​​镀后5天。看起来更先进的文化，与神经元扩展（小箭头），神经元簇（大箭头）和肌肉（中型箭头）。 <img src="/files/ftp_upload/2215/2215fig2.jpg" alt="图2" /> 图2。 dsRNAs治疗原发性细胞培养在384孔板 从账面Dmef2 - Gal4的胚胎，D42 - Gal4的，UAS的线粒体GFP转基因，其中Dmef2 - Gal4的D42 - Gal4的线粒体GFP的驱动器在肌肉和运动神经元的表达，分别为原代细胞中分离。针对要么lacZ基因（AC）或膨胀（如果）（DF）的dsRNAs细胞治疗。小学的肌肉和神经元可以看出，绿色荧光蛋白（A，C，D，F）。白色箭头指向神经扩展。鬼笔环肽染色显示肌动蛋白（箭头）在lacZ的dsRNAs（B和C）治疗文化的一个分支状的肌肉结构，但在文化的全面肌肉形态如果治疗dsRNAs（E和F）。在合并后的图像（C，F），肌肉都染成黄色，但红色的负面显示神经元及其扩展（白色箭头）。 <img src="/files/ftp_upload/2215/2215fig3.jpg" alt="图3" /> 图3。人类vitronectin涂盖玻片上培养的主要肌肉的共焦显微 主要肌肉immunofluorescently染色抗果蝇肌联蛋白（KZ）（红色）在合并和鬼笔环肽与肌动蛋白（在合并中的绿色）。顶部面板的野生型控制肌肉和底部的课后（CG31374）的RNAi缩短肌的肌肉。

<ol>
	<li>Ashburner, M., Roote, J., Sullivan, W., Ashburner, M., Hawley, 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=.">.</a> Laboratory culture of Drosophila. , 588-599 (1999).</li><li>Bai, J., Hartwig, J. H., Perrimon, N. S. A. L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=a+WH2-domain-containing+protein,+promotes+sarcomeric+actin+filament+elongation+from+pointed+ends+during+Drosophila+muscle+growth.">a WH2-domain-containing protein, promotes sarcomeric actin filament elongation from pointed ends during Drosophila muscle growth.</a> Dev. Cell. 13, 828-842 (2007).</li><li>Bai, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+interference+screening+in+Drosophila+primary+cells+for+genes+involved+in+muscle+assembly+and+maintenance+.">RNA interference screening in Drosophila primary cells for genes involved in muscle assembly and maintenance .</a> Development. 135, 1439-1449 (2008).</li><li>Sepp, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+neural+outgrowth+genes+using+genome-wide+RNAi.">Identification of neural outgrowth genes using genome-wide RNAi.</a> PloS. Genet. 4, e1000111-e1000111 (2008).</li><li>Donady, J. J., Fyrberg, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+culturing+of+Drosophila+embryonic+cells+in+vitro.">Mass culturing of Drosophila embryonic cells in vitro.</a> TCA Manual. 3, 685-687 (1977).</li><li>Seecof, R. L., Alleaume, N., Teplitz, R. L., Gerson, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+neurons+and+myocytes+in+cell+cultures+made+from+Drosophila+gastrulae.">Differentiation of neurons and myocytes in cell cultures made from Drosophila gastrulae.</a> Exp. Cell Res. 69, 161-173 (1971).</li><li>Bai, J., Sepp, K. J., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+of+Drosophila+primary+cells+dissociated+from+gastrula+embryos+and+their+use+in+RNAi+screening.">Culture of Drosophila primary cells dissociated from gastrula embryos and their use in RNAi screening.</a> Nature Protocols. 4, 1502-1512 (2009).</li></ol>

果蝇细胞的原代培养的发展模式可以相差很大，可能是由于在主细胞制备过程中的几个变量。首先，苍蝇产卵应该是年轻的（在一个星期内老）和健康（无病毒或细菌感染）。未受精或受感染的胚胎必须避免，因为他们是无用的，从这些胚胎来源的细胞不能区分，将只能存活1-2天。其次，在同质化过程中，重要的是不超载太多的胚胎匀浆。匀浆超载太多的胚胎，会导致碎片和死细胞，最终...

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<td>Shields and Sang M3 medium</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S3652</td>
<td>&nbsp;</td>
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<td>Fetal bovine serum</td>
<td>&nbsp;</td>
<td>JRH Biosciences</td>
<td>12103-500M</td>
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<td>Insulin from bovine pancreas</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>I-6634</td>
<td>&nbsp;</td>
<tr>
<td>Large embryo collection cages</td>
<td>&nbsp;</td>
<td>Genesee Scientific</td>
<td>59-101</td>
<td>&nbsp;</td>
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<td>#25 US standard testing sieve</td>
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<td>57334-454</td>
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<td>#45 US standard testing sieve</td>
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<td>#120 US standard testing sieve</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>57334-474</td>
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<td>Dounce tissue grinder, Wheaton 7 mL</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>62400-620</td>
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<td>Dounce tissue grinder,Kontes 40 mL</td>
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<td>Dounce tissue grinder,Kontes 40 mL</td>
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<td>Costar, 384-well plate</td>
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<td>VWR</td>
<td>29444-078</td>
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<td>Lab-Tek II Chambered coverglass</td>
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primary cell cultures from drosophila gastrula embryos

在这里，我们描述了一个准备从果蝇原肠期胚胎和培养原代细胞分离的方法。总之，大量上演来自年轻和健康的苍蝇的胚胎收集，消毒，然后身体成单细胞悬浮液，使用玻璃匀浆分离。镀合适的密度，在培养基中培养板或玻片上后，这些细胞可以进一步分化成几种形态不同的细胞类型，可以通过其特定的细胞标记鉴定。此外，我们目前的治疗与双链（DS）的RNA这些细胞，引起基因敲除的条件。在果蝇中的主要细胞的高效RNAi是通过简单地沐浴在培养基中含有双链RNA的细胞。开展有效的RNAi的扰动，与其他分子生物学，生物化学，细胞成像分析的能力，将允许在果蝇的主要细胞，尤其是那些涉及到分化的肌肉和神经细胞的回答各种各样的问题。

Watch this Scientific Journal Video about Primary Cell Cultures from Drosophila Gastrula Embryos at JoVE.com

Primary Cell Cultures from Drosophila Gastrula Embryos

我们提供一份详细的协议，准备原代细胞脱离<em&gt; Drosophil</em&gt;一个胚胎。能够进行有效的RNAi扰动，与其他分子生物学，生物化学和细胞成像方法，将允许各种问题加以解决，<em&gt;果蝇</em&gt;原代细胞。

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 ...

primary-cell-cultures-from-drosophila-gastrula-embryos

Research

JoVE Journal

Biology

Primary Cell Cultures from Drosophila Gastrula Embryos

13.8K 次观看.  Harvard Medical School. 我们提供一份详细的协议，准备原代细胞脱离 Drosophil一个胚胎。能够进行有效的RNAi扰动，与其他分子生物学，生物化学和细胞成像方法，将允许各种问题加以解决，果蝇原代细胞。

视频: Primary Cell Cultures from Drosophila Gastrula Embryos

Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the removal of brains from animals at 70-78 hrs after puparium formation. The isolated brains are shown after brief incubation in papain followed by several washes in serum-free growth medium. The process of mechanical dissociation of each brain in a 5 ul drop of media on a coverslip is illustrated. The axons and dendrites of the post-mitotic neurons are sheered off near the soma during dissociation but the neurons begin to regenerate processes within a few hours of plating. Images show live cultures at 2 days. Neurons continue to elaborate processes during the first week in culture. Specific neuronal populations can be identified in culture using GAL4 lines to drive tissue specific expression of fluorescent markers such as  GFP or RFP. Whole cell recordings have demonstrated the cultured neurons form functional, spontaneously active cholinergic and GABAergic synapses. A short video segment illustrates calcium dynamics in the cultured neurons using Fura-2 as a calcium indicator dye to monitor spontaneous calcium transients and nicotine evoked calcium responses in a dish of cultured neurons. These pupal brain cultures are a useful model system in which genetic and pharmacological tools can be used to identify intrinsic and extrinsic factors that influence formation and function of central synapses.

This video demonstrates the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. Views of live cultures show neurite outgrowth and imaging of calcium levels using Fura-2.

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 ...

科研

生物学

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 ...

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 ...

Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups of cells in a larger context are altered.  Watching how one cell interacts with and behaves in a unique environment is essential to characterizing cell functions.  Determining how the localized misexpression of a specific protein influences surrounding cells provides insightful information on the roles that protein plays in a variety of developmental processes.  Our lab uses the zebrafish model system to uniquely combine genetic approaches with classical transplantation techniques to generate genotypic or phenotypic chimeras.  We study neuron-glial cell interactions during the formation of forebrain commissures in zebrafish. This video describes a method that allows our lab to investigate the role of astroglial populations in the diencephalon and the roles of specific guidance cues that influence projecting axons as they cross the midline. Due to their transparency zebrafish embryos are ideal models for this type of ectopic cell placement or localized gene misexpression. Tracking transplanted cells can be accomplished using a vital dye or a transgenic fish line expressing a fluorescent protein. We demonstrate here how to prepare donor embryos with a vital dye tracer for transplantation, as well as how to extract and transplant cells from one gastrula staged embryo to another.  We present data showing ectopic GFP+ transgenic cells within the forebrain of zebrafish embryos and characterize the location of these cells with respect to forebrain commissures.  In addition, we show laser scanning confocal timelapse microscopy of Alexa 594 labeled cells transplanted into a GFP+ transgenic host embryo.  These data provide evidence that gastrula staged transplantation enables the targeted positioning of ectopic cells to address a variety of questions in Developmental Biology.

Zebrafish cell transplantation enables the combination of genetics and embryology to generate tissue specific chimeras. This video demonstrates gastrula staged cell transplantations that have allowed our lab to investigate the roles of astroglial populations and specific guidance cues during commissure formation in the forebrain.

组织有针对性的胚胎嵌合体：斑马鱼原肠胚细胞移植

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups ...

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 ...

Upright Imaging of Drosophila Embryos

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current techniques used to view such processes are somewhat limited. The following protocol describes a new technique for mounting fixed and labeled Drosophila embryos for coronal viewing with confocal imaging. This method consists of embedding embryos between two layers of glycerin jelly mounting media, and imaging jelly strips positioned upright. The first step for sandwiching the embryos is to make a thin bedding of glycerin jelly on a slide. Next, embryos are carefully aligned on this surface and covered with a second layer of jelly. After the second layer is solidified, strips of jelly are cut and flipped upright for imaging. Alternatives are described for visualizing the embryos depending upon the type of microscope stand to be used. Since all cells along the dorsal-ventral axis are imaged within a single confocal Z-plane, our method allows precise measurement and comparison of fluorescent signals without photobleaching or light scattering common to 3D reconstructions of longitudinally mounted embryos.

Upright Imaging of Drosophila Embryos

Here we present a mounting protocol for stained Drosophila embryos in an upright position that allows imaging of cross-sections using Confocal microscopy.

直立成像果蝇胚胎

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current ...

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

影像生活细胞的形状变化果蝇胚胎

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

kinesin的分离和纯化果蝇胚胎

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...

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 ...