作者要感谢约翰沃尔伍德这种方法的初步基础，TGK是支持由美国国立卫生研究院GM057483和AI076245和中国GM77073支持。

1。 7-47％蔗糖梯度制备<ol><li>准备蔗糖梯度有一天，在使用之前，让渐变成为连续。无菌7％和47％的蔗糖溶液，含50mm 的NH 4 CL，50毫米的Tris -醋酸的pH 值 7.0和12mm MgCl 2的和储存在4 ° C 1。 </li><li> 6梯度组合的7％和47％蔗糖的股票解决方案，使48毫升以下的蔗糖浓度。数码地面电视（1M股票）添加到每个蔗糖溶液至终浓度为1mm。 <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> 终浓度 </td><td> 7％的蔗糖</td><td> 47％的蔗糖 </td></tr><tr><td> 7％ </td><td> 48毫升</td><td> 0 </td></tr><tr><td> 17％ </td><td> 36毫升</td><td> 12毫升</td></tr><tr><td> 27％ </td><td> 24毫升</td><td> 24毫升</td></tr><tr><td> 37％ </td><td> 12毫升</td><td> 36毫升</td></tr><tr><td> 47％ </td><td> 0 </td><td> 48毫升</td></tr></tbody></table></li><li>为了倒梯度，巴斯德吸管附加保障，并用封口膜密封，20毫升注射器或使用长针。 7％的蔗糖添加7毫升1 × 3.5“polyallomer超速离心机试管底部，然后添加7毫升17％，7％的解决方案下的蔗糖溶液，放置巴斯德吸管试管的底部附近的一角，超过0.3吹打没有更快毫升/秒7毫升的27％，37％和47％的蔗糖溶液的重复，您应遵守线条清晰，层与层之间，最小的混合。商店梯度发生在4 ° C过夜连同转子，瓶将用于收获样品管。 </li></ol> 2。酵母提取物的制备<ol><li>增长酵母培养125毫升0.8-1.0外径600。添加酮的文化到终浓度为100微克/毫升，并继续摇动30 ° 15分钟的彗星。 </li><li>同时，准备裂解液中含有80微克/毫升放线菌酮，200微克/ ml肝素，0.2％DEPC，和10毫米的Tris - HCl pH值7.5，0.1M的氯化钠，氯化镁30MM 2。保持冰从这个角度上的一切。 </li><li>倒入一个250毫升离心瓶的文化，并填补了冰。在8000 XG（7250 RPM在SLA - 1500转子）离心5分钟在4 ° C洗涤沉淀一次用10毫升的冰冷的裂解液转移至15 mL聚丙烯管。在5900 XG（〜6000 RPM）在一个SLA - 600TC转子离心3分钟，在4 ° C重悬在0.5 mL裂解缓冲液，转移中的颗粒1.5ml离心管，加入0.5毫升冷冻，烘烤，酸洗玻璃珠。涡4个30秒的间隔，在冰上冷却管，相互之间间隔为30秒的细胞。添加到另一个裂解液0.5毫升1.5毫升管。在微量的Eppendorf 5417R离心（14,000 RPM）的最大速度离心10分钟在4 ° C上清转移到一个新的1.5 ml离心管。取出10μL到一个单独的管，以确定的OD 260 / OD 280比值（见下文步骤3.1）。商店提取物在-80 ° C </li></ol> 3。从哺乳动物细胞提取物的制备<ol><li>对于贴壁细胞，生长在100毫米的菜〜70％汇合。您将需要一个100毫米，每11毫升梯度菜（典型的产量是20 OD 260单位） 。金额较大或较小的梯度大小线性的规模。对于非贴壁细胞，约1 × 10 7细胞都需要每11毫升梯度。 </li><li>裂解之前，添加100μg/ mL的放线菌酮。在37 ° C下15分钟。传输板冰，在冰冷的PBS冲洗细胞两次。将来所有的步骤必须进行在0-4 ° C。 </li><li>的PBS吸删除所有痕迹（让板漏角度的冰床），并添加1 mL裂解缓冲液，刮去，并转移到预冷1.5 mL试管。裂解液成分，将需要的细胞类型和生长条件进行了优化。一个很好的起点缓冲区将20毫米的HEPES - KOH，pH值7.4，15毫米氯化镁2，氯化钾200毫米，1％海卫X - 100（V / V），100微克/毫升放线菌酮，2毫米的数码地面电视服务，和1 mg / ml肝素。的关键变量，镁和氯化钾，以及列入或排除非离子型洗涤剂，以提高提取的骨架或膜约束polysom​​es的浓度。通过两次通过27号注射器针头或5-10 dounce同质化的招用B型杵裂解细胞。应预冷前使用的注射器或匀浆。 </li><li>冷冻离心5分钟旋转14000 XG。将新鲜的裂解蔗糖梯度（哺乳动物细胞裂解缺乏Triton X - 100和肝素的缓冲，但在1.2和1.3所述，否则）或闪光灯以备后用液氮冻结。 </li></ol&gt; 4。渐变离心效果<ol><li>在冰上解冻polyribosome裂解液。保留10μL样品中加入1毫升水，并用分光光度计读取OD 260 / OD 280的比例确定核酸裂解液浓度。从酵母培养成长为描述一个典型的产量是50外径为单位。 </li><li>使用吸管尖端放在对管梯度表面附近的墙壁，轻轻层25 外径 260梯度顶部裂解单位。对于一个35 mL的梯度500μL裂解液通常是加载。可用于裂解缓冲液，以保证所有的梯度与同等体积加载。较低的数额的物质会产生更好的分辨率。使用镊子，小心地降低到一个水平转头桶的梯度。 </li><li>梯度离心4小时100000 XG在Surespin 630转子（23,000 RPM）在4 ° C。该协议可以适用于更小的体积的梯度。 </li></ol> 5。收集的数据和分数<ol><li>大约30分钟前完成4小时旋转针型号184管穿孔。将笔到网上ISCO型号UA - 5吸光度/荧光显示器。对吸光度的电源监控，以便分析样品前，要达到一个稳定的基准。电子收购的polysom​​e轮廓，可以很容易地获得通过附加一个DI - 148U的数据采集单元（DATAQ仪器）图表记录。配置文件可以实时收集和保存以供日​​后使用附带的WinDaq软件的注释。 </li><li>填充Fluorinert使用的逆向流动，迅速设置50毫升的注射泵。确保有没有气泡。从注射器管穿孔的软管连接，并暂时关闭3毫升/分钟的前进方向，以清除任何气泡软管Fluorinert流。 </li><li>放置一个废物管或烧杯系列如果收集馏分流动池下软管收集样品运行。 </li><li>小心取出polyallomer离心管中，含有蔗糖梯度离心转子，放在冰（前形成与冰空管的井，以避免干扰梯度）。 </li><li>为了分析细胞提取物，梯度管，管穿孔。管上方连接到电源插座和安全。提高阶段性底部的离心管，使针头穿透试管底部。一旦针头通过离心管底部的突出的前进方向，开始在6mL/min流Fluorinert。 </li><li>一旦Fluorinert已经开始流入管，监察运动的针上在线吸光度监测。一旦针开始移动，在图表上的运动，以60的速度设定。监视器将记录与40S核糖体亚基检测，并继续通过polyribosome络合物自由材料的外径254起。如果你正在收集RNA或蛋白质分析的分数，通常是0.2分的分数是（1毫升）。 RNA分析，分数应该是直接收集到含有Trizol试剂（Invitrogen公司），苯酚或SDS /蛋白酶K管上的首选方法提取而定。 </li><li>当年底的polyribosome轮廓已经达成，关闭流Fluorinert离心管和停止的吸光度/荧光显示器中的图表运动。 </li><li>缩回到注射器Fluorinert开始在相反的方向流动快速，请务必不要从蔗糖进入注射器管率从离心管。拧开管穿孔的离心管中，低针，取出管。 </li><li>重复步骤5.8 5.5每个蔗糖梯度。 </li><li>当所有的蔗糖梯度进行了分析，收回Fluorinert从软管进入注射器。删除每个分馏部分，包括在上方废物软管（管穿孔和流动池），并用清水冲洗干净。 </li></ol> 6。代表性的成果<img src="/files/ftp_upload/1948/1948fig1.jpg" alt="图1" /> 图1代表核糖体中提取的痕迹的酵母菌株BY4741中存在的放线菌酮（垫HIS3ΔLEU2Δ0 met15ΔΔ0 0 URA3）的准备。核糖体的提取物被分割利用7 47％的蔗糖梯度和分析，使用一台泵注射器仪器连接到紫外探测器。含有多聚核糖体80S，60S和40S核糖体的峰表示。

<ol>
	<li>Baim, S. B., Pietras, D. F., Eustice, D. C., Sherman, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mutation+allowing+an+mRNA+secondary+structure+diminishes+translation+of+Saccharomyces+cerevisiae+iso-1-cytochrome+C.">A mutation allowing an mRNA secondary structure diminishes translation of Saccharomyces cerevisiae iso-1-cytochrome C.</a> Mol. Cell. Biol. 5, 1839-1846 (1985).</li><li>Ripmaster, T. L., Vaughn, G. P., Woolford, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DRS1+to+DRS7,+novel+genes+required+for+ribosome+assembly+and+function+in+Saccharomyces+cerevisiae.">DRS1 to DRS7, novel genes required for ribosome assembly and function in Saccharomyces cerevisiae.</a> Mol. Cell. Biol. 13, 7901-7912 (1993).</li><li>Anand, M., Chakraburtty, K., Marton, M. J., Hinnebusch, A. G., Kinzy, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+interactions+between+yeast+translation+eukaryotic+elongation+factor+(eEF)+1A+and+eEF3.">Functional interactions between yeast translation eukaryotic elongation factor (eEF) 1A and eEF3.</a> J Biol Chem. 278, 6985-6991 (2003).</li><li>Serikawa, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transcriptome+and+its+translation+during+recovery+from+cell+cycle+arrest+in+Saccharomyces+cerevisiae.">The transcriptome and its translation during recovery from cell cycle arrest in Saccharomyces cerevisiae.</a> Mol Cell Proteomics. 2, 191-204 (2003).</li><li>Ingolia, N. T., Ghaemmaghami, S., Newman, J. R., Weissman, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+in+vivo+of+translation+with+nucleotide+resolution+using+ribosome+profiling.">Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling.</a> Science. 324, 218-223 (2009).</li><li>Valasek, L., Szamecz, B., Hinnebusch, A. G., Nielsen, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+stabilization+of+preinitiation+complexes+by+formaldehyde+cross-linking.">In vivo stabilization of preinitiation complexes by formaldehyde cross-linking.</a> Methods Enzymol. 429, 163-183 (2007).</li><li>Fletcher, J. E., Copeland, P. R., Driscoll, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polysome+distribution+of+phospholipid+hydroperoxide+glutathione+peroxidase+mRNA:+evidence+for+a+block+in+elongation+at+the+UGA/selenocysteine+codon.">Polysome distribution of phospholipid hydroperoxide glutathione peroxidase mRNA: evidence for a block in elongation at the UGA/selenocysteine codon.</a> Rna. 6, 1573-1584 (2000).</li><li>Ruan, H. J., Brown, C. Y., Morris, D. R., Richter, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Analysis of mRNA formation and function. , 305-321 (1997).</li><li>Martin, G. W., Berry, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selenocysteine+codons+decrease+polysome+association+on+endogenous+selenoprotein+mRNAs.">Selenocysteine codons decrease polysome association on endogenous selenoprotein mRNAs.</a> Genes Cells. 6, 121-129 (2001).</li><li>Lee, Y. Y., Cevallos, R. C., Jan, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+upstream+open+reading+frame+regulates+translation+of+GADD34+during+cellular+stresses+that+induce+eIF2alpha+phosphorylation.">An upstream open reading frame regulates translation of GADD34 during cellular stresses that induce eIF2alpha phosphorylation.</a> J Biol Chem. 284, 6661-6673 (2009).</li></ol>

从polyribosome配置文件中获得的信息，可以进入翻译状态的细胞提供有价值的见解。此外，可确定自己的核糖体亚基组装的状态2 。例如halfmers或80S和较大的山峰有轻微的肩膀上个人资料的权利的存在表明绑定的40S亚基等待60S亚基加入。执行在没有任何附加的放线菌酮或其他伸长抑制剂实验可以分析的流失率，这表明与否的伸长率是改变3。的分数本身是为一个特定的基因或蛋白质与核糖体的亚群，北方或西方的分数印迹协会随后决定的试剂的丰富来源。可确定的积极核糖体mRNA的总池通过相关的芯片 4或深度测序分析 5 。也可以稳定蛋白质协会由适当的另外一个交叉连接试剂6。 从哺乳动物细胞的Polyribosome分析也值得一提的，在不同的协议在建立所需的条件，以获得稳定的polysom​​es明显的困难。文献报道的缓冲区系统广泛变量7-10，从而有可能为细胞裂解液的具体优化的要求，或者是同样合理的缓冲系统，是不是只要与热切关注的重要新鲜裂解液在低温下保存。据我们所知，系统评价的影响哺乳动物polysom​​e形成的参数尚未见报道。

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<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>polyallomer ultracentrifuge tube</td>
<td>&nbsp;</td>
<td>Beckman</td>
<td>326823</td>
<td>1 x 3.5 in. (25x89mm)</td>
</tr>
<tr>
<td>Fluorinert</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>F9755</td>
<td>&nbsp;</td>
<tr>
<td>Cycloheximde</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C-7698</td>
<td>&nbsp;</td>
<tr>
<td>Heparin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H3393</td>
<td>&nbsp;</td>
<tr>
<td>BY4741</td>
<td>&nbsp;</td>
<td>Open Biosystems</td>
<td>YSC1048</td>
<td>Other strains work equally well</td>
</tr>		</tbody>
		</table>
		</div>

eukaryotic polyribosome profile analysis

蛋白质合成是一个复杂的细胞的过程，是多层次的监管。例如，全球翻译，可以抑制在开始阶段，或由多种氨基酸饥饿或生长因子撤出的细胞，如强调伸长阶段。另外，个别的mRNA的翻译，可调节mRNA的本地化或同源的microRNA存在。蛋白质合成的研究经常使用polyribosome的分析，阐明翻译调控机制或蛋白质合成的缺陷。在这个实验中，mRNA /核糖体的复合物是由真核细胞中分离出。蔗糖密度梯度分离势必被称为绑定到一个单一的核糖体或monosome的mRNA的多聚核糖体的多个核糖体mRNA的。分馏梯度允许不同的核糖体的人口及其相关的mRNA或蛋白质分离和量化。多聚核糖体的比例在规定条件下的差异，以monosomes可以在任的翻译起始或伸长/终止的缺陷指示。目前的mRNA在polyribosome分数的考试可以揭示是否对个人的mRNA队列被翻译与实验条件的变化。此外，核糖体的组装可以监测分析小核糖体大亚基梯度分离峰。在这段视频中，我们目前的原油从酵母细胞中，蔗糖梯度的提取分离和对结果的解释的核糖体中提取的准备方法。此过程是哺乳动物细胞容易适应。

Watch this Scientific Journal Video about Eukaryotic Polyribosome Profile Analysis at JoVE.com

Eukaryotic Polyribosome Profile Analysis

本文介绍了协议的真核细胞的核糖体翻译的提取。一旦提取，核糖体是由蔗糖梯度分馏分离到monosomes和多聚核糖体，允许不同的核糖体的人群进行分析。因此，这种方法是研究翻译调控的黄金标准。

真核Polyribosome剖面分析

Protein synthesis is a complex cellular process that is regulated at many levels. For example, global translation can be inhibited at the initiation phase ...

Research

JoVE Journal

Biology

52.4K 次观看.  University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School. 本文介绍了协议的真核细胞的核糖体翻译的提取。一旦提取，核糖体是由蔗糖梯度分馏分离到monosomes和多聚核糖体，允许不同的核糖体的人群进行分析。因此，这种方法是研究翻译调控的黄金标准。

视频: Eukaryotic Polyribosome Profile Analysis

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

一个神经元和星形胶质细胞共培养检测神经毒性的高含量分析

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

科研

生物学

Bioenergetic Profile Experiment using C2C12 Myoblast Cells

The ability to measure cellular metabolism and understand mitochondrial dysfunction, has enabled scientists worldwide to advance their research in understanding the role of mitochondrial function in obesity, diabetes, aging, cancer, cardiovascular function and safety toxicity.
Cellular metabolism is the process of substrate uptake, such as oxygen, glucose, fatty acids, and glutamine, and subsequent energy conversion through a series of enzymatically controlled oxidation and reduction reactions. These intracellular biochemical reactions result in the production of ATP, the release of heat and chemical byproducts, such as lactate and CO2 into the extracellular environment.
Valuable insight into the physiological state of cells, and the alteration of the state of those cells, can be gained through measuring the rate of oxygen consumed by the cells, an indicator of mitochondrial respiration - the Oxygen Consumption Rate - or OCR. Cells also generate ATP through glycolysis, i.e.: the conversion of glucose to lactate, independent of oxygen. In cultured wells, lactate is the primary source of protons. Measuring the lactic acid produced indirectly via protons released into the extracellular medium surrounding the cells, which causes acidification of the medium provides the Extra-Cellular Acidification Rate - or ECAR.
In this experiment, C2C12 myoblast cells are seeded at a given density in Seahorse cell culture plates. The basal oxygen consumption (OCR) and extracellular acidification (ECAR) rates are measured to establish baseline rates. The cells are then metabolically perturbed by three additions of different compounds (in succession) that shift the bioenergetic profile of the cell.
This assay is derived from a classic experiment to assess mitochondria and serves as a framework with which to build more complex experiments aimed at understanding both physiologic and pathophysiologic function of mitochondria and to predict the ability of cells to respond to stress and/or insults.

A description of a method for profiling mitochondrial function in cells is provided. The mitochondrial profile generated provides four parameters of mitochondrial function that can be measured in one experiment: basal respiration rate, ATP-linked respiration, proton leak, and reserve capacity.

使用C2C12成肌细胞的生物能简介实验

The ability to measure cellular metabolism and understand mitochondrial dysfunction, has enabled scientists worldwide to advance their research in ...

Correlative Light and Electron Microscopy (CLEM) as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets

The eukaryotic cell relies on complex, highly regulated, and functionally distinct membrane bound compartments that preserve a biochemical polarity necessary for proper cellular function. Understanding how the enzymes, proteins, and cytoskeletal components govern and maintain this biochemical segregation is therefore of paramount importance. The use of fluorescently tagged molecules to localize to and/or perturb subcellular compartments has yielded a wealth of knowledge and advanced our understanding of cellular regulation. Imaging techniques such as fluorescent and confocal microscopy make ascertaining the position of a fluorescently tagged small molecule relatively straightforward, however the resolution of very small structures is limited 1.
On the other hand, electron microscopy has revealed details of subcellular morphology at very high resolution, but its static nature makes it difficult to measure highly dynamic processes with precision 2,3. Thus, the combination of light microscopy with electron microscopy of the same sample, termed Correlative Light and Electron Microscopy (CLEM) 4,5, affords the dual advantages of ultrafast fluorescent imaging with the high-resolution of electron microscopy 6. This powerful technique has been implemented to study many aspects of cell biology 5,7. Since its inception, this procedure has increased our ability to distinguish subcellular architectures and morphologies at high resolution. 
Here, we present a streamlined method for performing rapid microinjection followed by CLEM (Fig. 1). The microinjection CLEM procedure can be used to introduce specific quantities of small molecules and/or proteins directly into the eukaryotic cell cytoplasm and study the effects from millimeter to multi-nanometer resolution (Fig. 2). The technique is based on microinjecting cells grown on laser etched glass gridded coverslips affixed to the bottom of live cell dishes and imaging with both confocal fluorescent and electron microscopy. Localization of the cell(s) of interest is facilitated by the grid pattern, which is easily transferred, along with the cells of interest, to the Epon resin used for immobilization of samples and sectioning prior to electron microscopy analysis (Fig. 3). Overlay of fluorescent and EM images allows the user to determine the subcellular localization as well as any morphological and/or ultrastructural changes induced by the microinjected molecule of interest (Fig. 4). This technique is amenable to time points ranging from &le;5 s up to several hours, depending on the nature of the microinjected sample.

Correlative Light and Electron Microscopy CLEM as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets

The CLEM technique has been adapted to analyze ultrastructural morphology of membranes, organelles, and subcellular structures affected by microinjected molecules. This method combines the powerful techniques of micromanipulation/microinjection, confocal fluorescent microscopy, and electron microscopy to allow millimeter to multi-nanometer resolution. This technique is amenable to a wide variety of applications.


The eukaryotic cell relies on complex, highly regulated, and functionally distinct membrane bound compartments that preserve a biochemical polarity ...

Establishment of Microbial Eukaryotic Enrichment Cultures from a Chemically Stratified Antarctic Lake and Assessment of Carbon Fixation Potential

Lake Bonney is one of numerous permanently ice-covered lakes located in the McMurdo Dry Valleys, Antarctica. The perennial ice cover maintains a chemically stratified water column and unlike other inland bodies of water, largely prevents external input of carbon and nutrients from streams. Biota are exposed to numerous environmental stresses, including year-round severe nutrient deficiency, low temperatures, extreme shade, hypersalinity, and 24-hour darkness during the winter 1. These extreme environmental conditions limit the biota in Lake Bonney almost exclusively to microorganisms 2.
Single-celled microbial eukaryotes (called "protists") are important players in global biogeochemical cycling 3 and play important ecological roles in the cycling of carbon in the dry valley lakes, occupying both primary and tertiary roles in the aquatic food web. In the dry valley aquatic food web, protists that fix inorganic carbon (autotrophy) are the major producers of organic carbon for organotrophic organisms 4, 2. Phagotrophic or heterotrophic protists capable of ingesting bacteria and smaller protists act as the top predators in the food web 5. Last, an unknown proportion of the protist population is capable of combined mixotrophic metabolism 6, 7. Mixotrophy in protists involves the ability to combine photosynthetic capability with phagotrophic ingestion of prey microorganisms. This form of mixotrophy differs from mixotrophic metabolism in bacterial species, which generally involves uptake dissolved carbon molecules. There are currently very few protist isolates from permanently ice-capped polar lakes, and studies of protist diversity and ecology in this extreme environment have been limited 8, 4, 9, 10, 5. A better understanding of protist metabolic versatility in the simple dry valley lake food web will aid in the development of models for the role of protists in the global carbon cycle.
We employed an enrichment culture approach to isolate potentially phototrophic and mixotrophic protists from Lake Bonney. Sampling depths in the water column were chosen based on the location of primary production maxima and protist phylogenetic diversity 4, 11, as well as variability in major abiotic factors affecting protist trophic modes: shallow sampling depths are limited for major nutrients, while deeper sampling depths are limited by light availability. In addition, lake water samples were supplemented with multiple types of growth media to promote the growth of a variety of phototrophic organisms.
RubisCO catalyzes the rate limiting step in the Calvin Benson Bassham (CBB) cycle, the major pathway by which autotrophic organisms fix inorganic carbon and provide organic carbon for higher trophic levels in aquatic and terrestrial food webs 12. In this study, we applied a radioisotope assay modified for filtered samples 13 to monitor maximum carboxylase activity as a proxy for carbon fixation potential and metabolic versatility in the Lake Bonney enrichment cultures.

Microbial eukaryotes are both a source of photosynthetically-derived carbon and top predatory species in permanently ice-covered Antarctic lakes. This report describes an enrichment culture approach to isolate metabolically versatile microbial eukaryotes from the Antarctic lake, Lake Bonney, and assesses inorganic carbon fixation potential using a radioisotope assay for Ribulose-1,5-bisphophate carboxylase oxygenase (RubisCO) activity.

建立真核微生物的富集培养从化学分层南极湖和固碳潜力评估

Lake Bonney is one of numerous permanently ice-covered lakes located in the McMurdo Dry Valleys, Antarctica. The perennial ice cover maintains a ...

Metabolic Profile Analysis of Zebrafish Embryos

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these relationships will help to understand the underlying etiology for a range of diseases linked with mitochondrial dysfunction, such as diabetes and obesity. Recent advances in instrumentation, has enabled the monitoring of distinct parameters of mitochondrial function in cell lines or tissue explants. Here we present a method for a rapid and sensitive analysis of mitochondrial function parameters in vivo during zebrafish embryonic development using the Seahorse bioscience XF 24 extracellular flux analyser. This protocol utilizes the Islet Capture microplates where a single embryo is placed in each well, allowing measurement of bioenergetics, including: (i) basal respiration; (ii) basal mitochondrial respiration (iii) mitochondrial respiration due to ATP turnover; (iv) mitochondrial uncoupled respiration or proton leak and (iv) maximum respiration. Using this approach embryonic zebrafish respiration parameters can be compared between wild type and genetically altered embryos (mutant, gene over-expression or gene knockdown) or those manipulated pharmacologically. It is anticipated that dissemination of this protocol will provide researchers with new tools to analyse the genetic basis of metabolic disorders in vivo in this relevant vertebrate animal model.

Zebrafish represent a powerful vertebrate model that has been under-utilised for metabolic studies. Here we describe a rapid way to measure the in vivo metabolic profile of developing zebrafish that allows the comparison of different mitochondrial function parameters between genetically or pharmacologically manipulated embryos, thereby increasing the applicability of this organism.

斑马鱼胚胎的代谢谱分析

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these ...

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and composed of mature enterocytes, goblet cells and enteroendocrine cells; and crypts, residing proximal to the submucosa and the muscularis, harboring adult stem and progenitor cells and mature Paneth cells, as well as stromal and immune cells of the crypt microenvironment. Until the last few years, in vitro studies of small intestine was limited to cell lines derived from either benign or malignant tumors, and did not represent the physiology of normal intestinal epithelia and the influence of the microenvironment in which they reside. Here, we demonstrate a method adapted from Sato et al. (2009) for culturing primary mouse intestinal crypt organoids derived from C57BL/6 mice. In addition, we present the use of crypt organoid cultures to assay the crypt metabolic profile in real time by measurement of basal oxygen consumption, glycolytic rate, ATP production and respiratory capacity. Organoids maintain properties defined by their source and retain aspects of their metabolic adaptation reflected by oxygen consumption and extracellular acidification rates. Real time metabolic studies in this crypt organoid culture system are a powerful tool to study crypt organoid energy metabolism, and how it can be modulated by nutritional and pharmacological factors.

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

Small intestinal crypt organoids cultured ex vivo provide a tissue culture system that recapitulates growth of crypts dependent on stem cells and their niche. We established a method to assay the metabolic profile in real time in primary mouse crypt organoids. We found organoids maintain physiological properties defined by their source.

代谢谱的实时分析<em&gt;离体</em&gt;鼠标肠隐窝类器官培养

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and ...

Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs contribute to genetic variation among somatic cells in multicellular organisms and to evolution of unicellular eukaryotes. Sensitive methods for detecting eccDNA are needed to clarify how these elements affect genome stability and how environmental and biological factors&#160;induce their formation in eukaryotic cells. This video presents a sensitive eccDNA-purification method called Circle-Seq. The method encompasses column purification of circular DNA, removal of remaining linear chromosomal DNA, rolling-circle amplification of eccDNA, deep sequencing, and mapping. Extensive exonuclease treatment&#160;was&#160;required for sufficient linear chromosomal DNA degradation. The rolling-circle amplification step by &#966;29 polymerase enriched for circular DNA over linear DNA. Validation of the Circle-Seq method on three S. cerevisiae CEN.PK populations of 1010 cells detected hundreds of eccDNA profiles in sizes larger than 1 kilobase. Repeated findings of ASP3-1, COS111, CUP1, RSC30,&#160;HXT6, HXT7 genes on circular DNA in both S288c and CEN.PK suggests that DNA circularization is conserved between strains at these loci. In sum, the Circle-Seq method has broad applicability for genome-scale screening for eccDNA in eukaryotes as well as for detecting specific eccDNA types.

This paper presents a sensitive method called Circle-Seq for purifying extrachromosomal circular DNA (eccDNA). The method encompasses column purification, removal of remaining linear chromosomal DNA, rolling-circle amplification and high-throughput sequencing. Circle-Seq is applicable to genome-scale screening of eukaryotic eccDNA and studying genome instability and copy-number variation.

从真核细胞染色体外的环状DNA基因组范围内的净化

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs ...

Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the invasion of non-indigenous species. However, an exact identification of field predator-prey interactions is difficult in many cases. These analyses are often based on a visual evaluation of gut content or the analysis of stable isotope ratios (&#948;15N and &#948;13C). Such methods require comprehensive knowledge about, respectively, morphologic diversity or isotopic signature from individual prey organisms, leading to obstacles in the exact identification of prey organisms. Visual gut content analyses especially underestimate soft bodied prey organisms, because maceration, ingestion and digestion of prey organisms make identification of specific species difficult. Hence, polymerase chain reaction (PCR) based strategies, for example the use of group-specific primer sets, provide a powerful tool for the investigation of food web interactions. Here, we describe detailed protocols to investigate the gut contents of macroinvertebrate consumers from the field using group-specific primer sets for nuclear ribosomal deoxyribonucleic acid (rDNA). DNA can be extracted either from whole specimens (in the case of small taxa) or out of gut contents of specimens collected in the field. Presence and functional efficiency of the DNA templates need to be confirmed directly from the tested individual using universal primer sets targeting the respective subunit of DNA. We also demonstrate that consumed prey can be determined further down to species level via PCR with unmodified group-specific primers combined with subsequent single strand conformation polymorphism (SSCP) analyses using polyacrylamide gels. Furthermore, we show that the use of different fluorescent dyes as labels enables parallel screening for DNA fragments of different prey groups from multiple gut content samples via automated fragment analysis.

Most common gut content analyses of macroinvertebrates are visual. Requiring intense knowledge about morphological diversity of prey organisms, they miss soft bodied prey and, due to strong comminution of prey, are nearly impossible for some organisms, including amphipods. We provide detailed, novel genetic approaches for macroinvertebrate prey identification in the diet of amphipods.

用基团特异 rDNA 引物对水生无脊椎动物进行遗传肠道含量分析的实验室规程

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the ...

Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy (SEM)

Scanning electron microscopy (SEM) is a widely available technique that has been applied to study biological specimens ranging from individual proteins to cells, tissues, organelles, and even whole organisms. This protocol focuses on two chemical drying methods, hexamethyldisilazane (HMDS) and t-butyl alcohol (TBA), and their application to imaging of both prokaryotic and eukaryotic organisms using SEM. In this article, we describe how to fix, wash, dehydrate, dry, mount, sputter coat, and image three types of organisms: cyanobacteria (Toxifilum mysidocida, Golenkina sp., and an unknown sp.), two euglenoids from the genus Monomorphina (M. aenigmatica and M. pseudopyrum), and the fruit fly (Drosophila melanogaster). The purpose of this protocol is to describe a fast, inexpensive, and simple method to obtain detailed information about the structure, size, and surface characteristics of specimens that can be broadly applied to a large range of organisms for morphological assessment. Successful completion of this protocol will allow others to use SEM to visualize samples by applying these techniques to their system.

Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy SEM

SEM analysis is an effective method to aid in species identification or phenotypic discrimination. This protocol describes the methods for examining specific morphological details of three representative types of organisms and would be broadly applicable to examining features of many organismal and tissue types.

用化学干燥进行扫描电镜 (sem) 形态分析的原核生物和真核生物的制备

Scanning electron microscopy (SEM) is a widely available technique that has been applied to study biological specimens ranging from individual proteins to ...

Image Rendering Techniques in Postmortem Computed Tomography: Evaluation of Biological Health and Profile in Stranded Cetaceans

With 6 years of experience in implementing virtopsy routinely into the Hong Kong cetacean stranding response program, standardized virtopsy procedures, postmortem computed tomography (PMCT) acquisition, postprocessing, and evaluation were successfully established. In this pioneer cetacean virtopsy stranding response program, PMCT was performed on 193 stranded cetaceans, providing postmortem findings to aid necropsy and shed light on the biological health and profile of the animals. This study aimed to assess 8 image rendering techniques in PMCT, including multiplanar reconstruction, curved planar reformation, maximum intensity projection, minimum intensity projection, direct volume rendering, segmentation, transfer function, and perspective volume rendering. Illustrated with practical examples, these techniques were able to identify most of the PM findings in stranded cetaceans and served as a tool to investigate their biological health and profile. This study could guide radiologists, clinicians and veterinarians through the often difficult and complicated realm of PMCT image rendering and reviewing.

The Hong Kong cetacean stranding response program has incorporated postmortem computed tomography, which provides valuable information on the biological health and profile of the deceased animals. This study describes 8 image rendering techniques that are essential for the identification and visualization of postmortem findings in stranded cetaceans, which will help clinicians, veterinarians and stranding response personnel worldwide to fully utilize the radiological modality.

死后计算断层扫描中的图像渲染技术：受困鲸目动物的生物健康和特征评估

With 6 years of experience in implementing virtopsy routinely into the Hong Kong cetacean stranding response program, standardized virtopsy procedures, ...