Name: bromodeoxyuridine (brdu) labeling and subsequent fluorescence activated cell sorting for culture-independent identification of dissolved organic carbon-degrading bacterioplankton
Uploaded: 2011-09-10T14:00:00
Description: Watch this Scientific Journal Video about Bromodeoxyuridine BrdU Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degradin

这个项目的经费是由国家科学基金会资助OCE1029607（XM）和MCB0702125（MAM），戈登和贝蒂摩尔基金会（MAM）的。

1。水样加工<ol><li>通过1微米孔径的过滤膜过​​滤器10L环境用水，以去除大颗粒和bacteriovores。水滤液收集在一个carboy。 </li><li>转让36毫升滤液每到3无菌Eppendorf管中含有4毫升新鲜配制的多聚甲醛溶液（PFA 10％）（50毫升）。在室温下孵育2小时，以保持细胞。收集到0.22微米孔径的白色膜过滤器真空过滤的细胞。通过真空过滤的过滤器10毫升磷酸盐缓冲液（PBS）洗过滤器。标签作为阴性对照的过滤...

<ol>
	<li>Pernthaler, A., Pernthaler, J., Schattenhofer, M., Amann, R. <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+DNA-synthesizing+bacterial+cells+in+coastal+North+Sea+plankton.">Identification of DNA-synthesizing bacterial cells in coastal North Sea plankton.</a> Appl. Environ. Microbiol. 68, 5728-5728 (2002).</li><li>Urbach, E., Vergin, K. L., Giovannoni, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunochemical+detection+and+isolation+of+DNA+from+metabolically+active+bacteria.">Immunochemical detection and isolation of DNA from metabolically active bacteria.</a> Appl. Environ. Microbiol. 65, 1207-12 (1999).</li><li>Mou, X. Z., Hodson, R. E., Moran, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterioplankton+assemblages+transforming+dissolved+organic+compounds+in+coastal+seawater.">Bacterioplankton assemblages transforming dissolved organic compounds in coastal seawater.</a> Environ. Microbiol. 9, 2025-2025 (2007).</li><li>Hodson, R. E., Dustman, W. A., Garg, R. P., Moran, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+PCR+for+visualization+of+microscale+distribution+of+specific+genes+and+gene+products+in+prokaryotic+communities.">In situ PCR for visualization of microscale distribution of specific genes and gene products in prokaryotic communities.</a> Appl. Environ. Microbiol. 61, 4074-4074 (1995).</li><li>Dinjens, W. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bromodeoxyuridine+(BrdU)+immunocytochemistry+by+exonuclease+III+(Exo+III)+digestion.">Bromodeoxyuridine (BrdU) immunocytochemistry by exonuclease III (Exo III) digestion.</a> Histochemistry. 98, 199-199 (1992).</li><li>Kirchman, D. L., Yu, L. Y., Fuchs, B. M., Amann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+bacterial+communities+in+aquatic+systems+as+revealed+by+filter+PCR.">Structure of bacterial communities in aquatic systems as revealed by filter PCR.</a> Aquat. Microb. Ecol. 26, 13-13 (2001).</li><li>Delong, E. F., Wickham, G. S., Pace, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylogenetic+stains:+ribosomal+RNA-based+probes+for+the+identification+of+single+cells.">Phylogenetic stains: ribosomal RNA-based probes for the identification of single cells.</a> Science. 243, 1360-1360 (1989).</li><li>Artursson, V., Jansson, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+bromodeoxyuridine+immunocapture+to+identify+active+bacteria+associated+with+arbuscular+mycorrhizal+hyphae.">Use of bromodeoxyuridine immunocapture to identify active bacteria associated with arbuscular mycorrhizal hyphae.</a> Appl. Environ. Microbiol. 69, 6208-6208 (2003).</li><li>Mou, X. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+carbon+processing+by+generalist+species+in+the+coastal+ocean.">Bacterial carbon processing by generalist species in the coastal ocean.</a> Nature. 451, 708-708 (2008).</li><li>Mou, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow-cytometric+cell+sorting+and+subsequent+molecular+analyses+for+culture-independent+identification+of+bacterioplankton+involved+in+dimethylsulfoniopropionate+transformations.">Flow-cytometric cell sorting and subsequent molecular analyses for culture-independent identification of bacterioplankton involved in dimethylsulfoniopropionate transformations.</a> Appl. Environ. Microbiol. 71, 1405-1405 (2005).</li><li>Dean, F. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+human+genome+amplification+using+multiple+displacement+amplification.">Comprehensive human genome amplification using multiple displacement amplification.</a> Proc. Natl. Acad. Sci. U.S.A. 99, 5261-5261 (2002).</li></ol>

我们的做法夫妇BrdU掺入，流式细胞仪和16S rDNA序列分析，让物种一级的浮游细菌的代谢水环境中的个人的DOC组件的识别。 BrdU掺入检测标签的细菌细胞的代谢活动，只允许对活性细菌的分析，因此不包括休眠细胞。在我们的方法， 在原位 immunodetected和细菌有不同层次的BrdU掺入BrdU掺入随后可视化，使用流式细胞仪进行分组和排序。的BrdU纳入细胞的分析也有报道使用的BrdU标记的DNA磁珠immu...

<table border="1"><tbody><tr><td> 名称 </td><td> 类型 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> BrdU </td><td>试剂</td><td>西格玛</td><td> B5002 - 5G </td><td></td></tr><tr><td>溶菌酶</td><td>试剂</td><td>西格玛</td><td> L6876 - 5G </td><td></td></tr><tr><td>蛋白酶K </td><td>试剂</td><td>西格玛</td><td> P2308 - 25毫克</td><td></td></tr><tr><td>原位细胞增殖试剂盒，FLUOS </td><td>套件</td><td>罗氏公司</td><td> 11810740001 </td><td>抗BrdU FLUOS和反应比制造商建议的孵化每缓冲区消费。 </td></tr><tr><td>帧密封孵化钱伯斯</td><td>材料</td><td> Bio - Rad公司</td><td> SLF - 1201 </td><td></td></tr><tr><td>聚碳酸酯膜过滤器（直径142毫米，1.0微米孔径） </td><td>材料</td><td> Millipore公司</td><td> FALP14250 </td><td></td></tr><tr><td>聚碳酸酯膜过滤器（直径25毫米，0.2微米孔径） </td><td>材料</td><td> Millipore公司</td><td> FGLP02500 </td><td></td></tr><tr><td>插图PuReTaq现成的PCR珠</td><td>套件</td><td> GE医疗集团</td><td> 27-9559-01 </td><td></td></tr><tr><td> QIAquick凝胶提取试剂盒</td><td>套件</td><td> QIAGEN公司</td><td> 28704 </td><td></td></tr><tr><td>防故障PCR检测系统</td><td>套件</td><td>震中</td><td> FS99060 </td><td></td></tr></tbody></table>

bromodeoxyuridine (brdu) labeling and subsequent fluorescence activated cell sorting for culture-independent identification of dissolved organic carbon-degrading bacterioplankton

微生物介导许多​​溶解的有机碳（DOC）基板在水生环境退化的主要代理商。然而，鉴定，改造大自然的DOC的具体池的细菌类群构成一个技术上的挑战。 在这里，我们描述了一个方法，夫妇溴脱氧尿嘧啶核苷（BrdU）掺入，荧光激活细胞分选（FACS），和16S rRNA基因的分子分析功能，使文化有辱人格的特定DOC的化合物在水生环境的浮游细菌能够独立识别。设置为接收BrdU和模型DOC化合物（DOC修订），或只BrdU（不加控制）一式三份浮游细菌的缩影。 BrdU替代品在新合成的细菌的DNA和BrdU标记的DNA的胸苷的位置可以随时immunodetected 1,2。通过一个24小时孵化，浮游细菌，可以使用添加的DOC复合预计将有选择地激活，因此有较高水平的BrdU掺入比在DOC的修订和细胞非反应细胞（您好细胞）在无除了控制（低BrdU掺入细胞，李细胞）。荧光免疫检测后，您好细胞区分和身体从李细胞分离，荧光激活细胞分选（FACS）3 。排序DOC - 反应的细胞（您好细胞）的DNA提取和分类学上通过随后的16S rRNA基因为基础的分析，包括PCR，克隆库的建设和测序鉴定。

Watch this Scientific Journal Video about Bromodeoxyuridine BrdU Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degradin

Bromodeoxyuridine BrdU Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degrading Bacterioplankton

环境浮游细菌培养与溶解有机碳（DOC）的化合物和一个DNA标记试剂，溴脱氧尿嘧啶核苷（BrdU）的一种模式。随后，商务部降解细胞分离升高BrdU使用荧光激活细胞分选（FACS）纳入散装社会。随后的分子分析这些细胞，然后确定。

溴脱氧尿嘧啶核苷（BrdU）标记和文化独立的鉴定溶解有机随后的荧光激活细胞分选碳有辱人格的浮游细菌

Microbes are major agents mediating the degradation of numerous dissolved organic carbon (DOC) substrates in aquatic environments. However, identification ...

Research

JoVE Journal

Biology

Bromodeoxyuridine (BrdU) Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degrading Bacterioplankton

Generation of Human CD40-activated B cells

人CD40激活的B细胞的生成

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

科研

生物学

Fluorescence Activated Cell Sorting of Plant Protoplasts

植物原生质体的荧光激活细胞分选

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental ...

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

脂质囊泡介导的亲和层析法，使用磁性细胞分选（LIMACS）：一种新的方法来分析蛋白质 - 脂质相互作用

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

细胞特异性分析<em&gt;拟南芥</em&gt;叶用荧光激活细胞分选

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

优化细胞分裂监测细胞示踪染料的染色和扩散建模方法

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting (FACS)

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting FACS

新鲜组织分离正常和癌症相关的成纤维细胞的荧光激活细胞分选（FACS）

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their ...

Sorting of Streptomyces Cell Pellets Using a Complex Object Parametric Analyzer and Sorter

Sorting of Streptomyces Cell Pellets Using a Complex Object Parametric Analyzer and Sorter

排序<em&gt;链霉菌</em&gt;细胞沉淀用一个复杂的对象参数分析仪和分选机

Streptomycetes are filamentous soil bacteria that are used in industry for the production of enzymes and antibiotics. When grown in bioreactors, these ...

Nanogold Labeling of the Yeast Endosomal System for Ultrastructural Analyses

酵母核内体系统的超微结构分析的纳米金标记

Endosomes are one of the major membrane sorting checkpoints in eukaryotic cells and they regulate recycling or destruction of proteins mostly from the ...

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

核酸，蛋白质与甲基转移和辅酶类似物序列特异性标签

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

从兔模型中分离纯化滑液源性间充质干细胞的磁活化细胞分选策略

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...