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器，并把它们存放在-20 ° C </li></ol> 步骤1.3-1.4建立DOC -有限的条件下是可选的。 <ol start="3"><li>添加（5微米的NH 4 CL，5微米NANO 3，和1μM的NaH 2 PO 4决赛中浓度）到carboy无机氮和磷的混合物。 </li><li> 在现场温度孵育偶尔鼓动48小时在黑暗中。 </li></ol> 2。建立和孵化缩影<ol><li>装满800毫升水样从第1.4步建立的缩影carboy每6个1 - L玻璃烧瓶。加入BrdU，终浓度为（10微米），每个缩影。拌匀。 </li><li>添加1毫升模型DOC复合解决方案分为三个缩影，这些将作为商务部修订。 1毫升无菌PBS添加到剩下的三个缩影，这些将作为无除了控制。 </li><li>在一个孵化器中孵化的缩影和孵化在现场温度在黑暗中晃动，而在100转。 </li><li>从每一个缩影和转让到50毫升无菌Eppendorf公司在时间点0，8，16，和24小时的管收集36毫升的水样。立即加入4毫升新鲜煤灰（10％）收集管和孵育2小时在室温下保存的细胞。 </li><li>通过0.22微米孔径的聚碳酸酯膜过滤器过滤器保存的细胞。用10 ml PBS冲洗过滤器。立即着手下一步或存放于-20 ° C的过滤器</li></ol> 在原位免疫检测BrdU掺入3。 <ol><li>在室温下解冻的过滤器（商务部修订的样品，不加控制和阴性对照）。 </li><li>应用溶菌酶溶液1毫升10毫克/毫升溶菌酶蛋清在100毫米，50纳米EDTA的Tris（PH = 8）] 4覆盖过滤器上的细菌细胞。在室温下孵育30分钟。通过下吸10毫升PBS冲洗过滤器。 </li><li>添加蛋白酶K溶液1毫升[2 mg / ml的蛋白酶K在100毫米，50纳米EDTA的Tris（PH = 8）] 4覆盖过滤器上的细菌细胞。在室温下孵育30分钟。通过下吸10毫升PBS冲洗过滤器。 </li><li>加入1毫升的核酸外切酶的解决方案[核酸外切酶III（50 U / ml）的氯化镁 2 5毫米和50毫米的Tris - HCl] 5到过滤器上的细菌细胞。 37 ° 30分钟。通过下吸10毫升PBS冲洗过滤器。 </li><li>根据制造商的指示组装框架密封孵化室（BIO - RAD）。 </li><li>酒精清洁干燥的表面上使用无菌刀片切成八分过滤器。 </li><li>使用镊子，放入一个组装框架密封孵化室（每个过滤器样品需要8帧密封腔）第八部分的过滤器。过滤器部分包含单元格方应朝上。过滤器的背面的水分，将允许过滤棒的幻灯片。如果过滤器变得干燥，适用于DIH 2澳商会中心在幻灯片上的过滤器部分一个小滴（2μL）。 </li></ol> 步骤3.8-3.20使用罗氏原位细胞增殖试剂盒的试剂，FLUOS从制造商的指示修改的程序。 除对PBS，所有的试剂套件内提供。 <ol start="8"><li>施加足够的孵育缓冲液（0.5％，0.1％Tween20的PBS，牛血清蛋白），均匀地覆盖整个孵化室的过滤器部分，而不会造成溢出，一旦密封。 </li><li>聚酯放置在孵化室帧帧盖。按上下紧紧密封的孵化室。以上的过滤器，避免气泡。为在室温下在黑暗中10分钟孵育密封腔。 </li><li>删除聚酯密封，打开孵化室。 （注:开放的商会，有时会拉起来的帧从幻灯片密封在这种情况下，准备一个新腔以下步骤。） </li><li>吸取了孵化室的一个角落缓冲区。避免接触过滤器。 </li><li>轻轻吹打100μLPBS冲洗过滤器和从会议厅出来的3倍。 </li><li>准备抗BrdU - FLUOS以下步骤使用前制造商建议的工作解决方案。 </li><li>吸取120μL抗BrdU FLUOS工作到过滤器的解决方案，照顾到均匀地覆盖整个表面。 </li><li>与一个新的聚酯盖重新密封腔。避免过滤器顶部的气泡。室在黑暗中在37 ° C为3小时。这一步将在原地异硫氰酸荧光素（FITC）标记BrdU纳入DNA。 </li><li>删除聚酯盖，打开孵化室。吸取了抗BrdU - FLUOS工作液。用PBS清洗过滤器的3倍。 </li><li>将过滤器从孵化室，无菌的表面。切片成小块用消毒的刀片的过滤器"部分。 </li><li>转移到2 ml离心管，每片含1毫升PBS的滤波器件。紧瓶盖的封口膜管和密封。在37 ° C和10分钟200转。 </li><li>安全和最高速度为5分钟到一个vortexer涡管。吸取上清液到15毫升无菌Eppendorf管。重复5次以上的孵化和涡步骤。结合在同一15 ml Eppendorf管中，每个样品的上清液。通常情况下，80％的细胞可以恢复暂停。 </li><li>商店取上清液，重悬细胞在4 ° C。 2天之内进行排序。 </li></ol> 4。流式细胞仪分析 这里描述的是一个BD FACSAria流式细胞仪和相应的软件程序。 <ol><li>优化制造商所建议的流式细胞仪以下步骤设置。这就要求:调整放大控制，设置所需的片断参数和甜蜜点;优化实验鞘压力的激光延迟和面积换算因子，FSC和SSC，FSC的阈值电压，FSC荧光缩放，荧光光电倍增管电压优化设置等</li><li>基于FITC和侧散射（SSC）的荧光强度流式细胞仪（FCM）上运行的阴性对照样品。增加的FITC标记的阈值，直到阴性对照无细胞可以FITC - SSC采集显示的可视化。 </li><li>运行不加控制样品和研究万细胞FITC和SSC收购的分布格局。定义一个门，附上所有的细胞，并指定为"低强度的细胞"（LIS）。 </li><li>运行DOC修订的样品和研究基于FITC和SSC万细胞的分布格局。有些细胞会出现在预设的李门。定义另一个门附上的细胞，具有较高的荧光强度比LIS（他）。获得统计数据，以查看门细胞的相对丰度。 </li><li>排序您好细胞，成集合，其中包含500μLPBS管"净化1滴"模式。终止排序时，他的人数达50万计数。 </li></ol> 5。筛选PCR扩增16S rRNA基因 过滤PCR程序修改Kirchman等。 <ol><li>筛选到一个白色的排序细胞，0.22微米孔径，25毫米直径，聚碳酸酯滤膜过滤。剪掉的过滤器，不包含任何使用无菌刀片的细菌细胞的边缘。切成8个同等大小的块过滤器。 </li><li>到PCR反应管中放置一个单一的过滤片，与细胞面临着外来的管。 PCR反应管中加入45μLPCR级水。淹没的过滤器完全在水中。 </li><li> 2罗氏插图PuReTaq阅读到进入PCR反应管PCR珠，简要旋涡。添加正向和反向的16S rRNA基因的引物（0.4微米每个引物终浓度），如27F和1492R 7各2μL，每个PCR反应管中。 </li><li> （可选）添加加入1μl牛血清白蛋白溶液（BSA，终浓度是30μg/100μL）的PCR反应，以帮助吸附放大抑制剂。如果选择不添加牛血清白蛋白，加水加入1μl。 </li><li>进行PCR扩增热循环仪上一个。一个触摸下来PCR程序的建议，其中的退火温度，比上一季度减少62至52 ° C时为1 ° C，每循环11周期由15个循环，退火温度52 ° C。所有循环包括变性（95℃），退火（62至52℃）和扩展（72℃）50年代的持续时间步骤。 PCR程序也包括在最初的3分钟变性和最后的10分钟延长步骤。 </li><li>溴化乙锭染色的1％琼脂糖凝胶电泳，确认PCR扩增。海关从凝胶中的PCR扩增和凯杰QIAquick凝胶提取试剂盒的清洁。 </li><li>每个样本执行两个额外的PCR扩增，每次使用一个新的过滤器"部分。经过PCR凝胶purificati上，池同一样品的扩增产物。纯净16S rDNA的扩增产物，现在准备允许分类鉴定，如克隆库的建设和测序，分子生物学分析。 </li></ol> 6。代表性的成果: 代表使用为例，腐胺降解菌的研究结果说明。从格鲁吉亚沿海现场采集水样，并按照上述程序进行处理。流式细胞仪分析显示，腐胺除了诱发高FITC荧光强度的细菌集团的发展，说明高BrdU掺入率（图1）。这些细胞被指定为高BrdU掺入细胞（他），预计包含大多为腐胺降解菌。他在不加控制，只包含与下级BrdU掺入（LIS）的细胞缺少。 LIS，预计主要包括浮游细菌，无法使用添加腐胺。他整理成分离的导管，然后到膜过滤器收集。获得高您好使用过滤器PCR细胞16S rRNA基因扩增产物。 <img src="/files/ftp_upload/2855/2855fig1.jpg" alt="图1"/> 图1。流量没有除了控制和孵化24小时后收集的样品模型化合物的修订（腐胺作为一个例子）流式细胞仪分析。细胞分布的分析是基于（1）的FITC标记（X轴），这是正相关，BrdU掺入水平的荧光强度，（2）侧散射（SSC，Y轴），这是正相关的细胞大小。门符号是基于BrdU掺入，（高，高BrdU掺入，李，低BrdU的掺入）的水平。 HI和李细胞相对百分比显示在相应的门。

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	<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>

没有利益冲突的声明。

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 ...

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Biology

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

20.9K 次观看.  Kent State University. 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。

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

Measuring Exocytosis in Neurons Using FM Labeling

The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission.  Here we used real-time imaging of vesicles labeled with FM dye to monitor the rate of presynaptic vesicle release.  FM4-64 is a red fluorescent amphiphilic styryl dye that embeds into the membranes of synaptic vesicles as endocytosis is stimulated.  Lipophilic interactions cause the dye to greatly increase in fluorescence, thus emitting a bright signal when associated with vesicles and a nominal one when in the extracellular fluid. After a wash step is used to help remove external dye within the plasma membrane, the remaining FM is concentrated within the vesicles and is then expelled when exocytosis is induced by another round of electrical stimulation. The rate of vesicles release is measured from the resulting decrease in fluorescence.  Since FM dye can be applied external and transiently, it is a useful tool for determining rates of exocytosis in neuronal cultures, especially when comparing the rates between transfected synapses and neighboring control boutons.



The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission. Here we used real-time imaging of vesicles labeled with the red fluorescent dye FM 4-64 to measure the rate of presynaptic vesicle release in hippocampal neuronal cultures.

测量中使用FM标签的神经元的胞外分泌

The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission.  Here we used real-time ...

科研

生物学

Generation of Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. 
In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). 
After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime na&#970;ve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

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 ...

Fluorescence Activated Cell Sorting of Plant Protoplasts

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis.
An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: PSCR::GFP (endodermis and quiescent center) and PWOX5::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution.
FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time.
This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce cell types (e.g. quiescent center cells). Lastly, a growth setup for Arabidopsis seedlings is demonstrated that enables uncomplicated treatment of the plants prior to cell sorting (e.g. for the cell type-specific analysis of biotic or abiotic stress responses). Potential supplementary uses for FACS of plant protoplasts are discussed.

A method for isolating specific cell types from plant material is demonstrated. This technique employs transgenic marker lines expressing fluorescent proteins in particular cell types, cellular dissociation and Fluorescence Activated Cell Sorting. Additionally, a growth setup is established here that facilitates treatment of Arabidopsis thaliana seedlings prior to cell sorting.

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

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids.
 To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. 
 We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane. Additional lipid protein complexes can be identified using proteomics analysis of lipid binding protein co-purified with the lipid vesicles.

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

To test the interaction of a protein with its target lipid we used MACS and Annexin V-conjugated magnetic beads and lipid vesicles synthesized from the target lipid and Annexin V-binding phosphatidylserine. Proteins bound to the target lipid are co-purified and analyzed after elution from the beads.

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

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the Arabidopsis leaf is a complex organ made up of many different cell types and several structures. At the same time, the growing leaf contains cells at different stages of development, with the cells furthest from the petiole being the first to stop expanding and undergo senescence1. Different cells within the leaf are therefore dividing, elongating or differentiating; active, stressed or dead; and/or responding to stimuli in sub-sets of their cellular type at any one time. This makes genomic study of the leaf challenging: for example when analyzing expression data from whole leaves, signals from genetic networks operating in distinct cellular response zones or cell types will be confounded, resulting in an inaccurate profile being generated.
To address this, several methods have been described which enable studies of cell specific gene expression. These include laser-capture microdissection (LCM)2 or GFP expressing plants used for protoplast generation and subsequent fluorescence activated cell sorting (FACS)3,4, the recently described INTACT system for nuclear precipitation5 and immunoprecipitation of polysomes6.
FACS has been successfully used for a number of studies, including showing that the cell identity and distance from the root tip had a significant effect on the expression profiles of a large number of genes3,7. FACS of GFP lines have also been used to demonstrate cell-specific transcriptional regulation during root nitrogen responses and lateral root development8, salt stress9 auxin distribution in the root10 and to create a gene expression map of the Arabidopsis shoot apical meristem11. Although FACS has previously been used to sort Arabidopsis leaf derived protoplasts based on autofluorescence12,13, so far the use of FACS on Arabidopsis lines expressing GFP in the leaves has been very limited4. In the following protocol we describe a method for obtaining Arabidopsis leaf protoplasts that are compatible with FACS while minimizing the impact of the protoplast generation regime. We demonstrate the method using the KC464 Arabidopsis line, which express GFP in the adaxial epidermis14, the KC274 line, which express GFP in the vascular tissue14 and the TP382 Arabidopsis line, which express a double GFP construct linked to a nuclear localization signal in the guard cells (data not shown; Figure 2). We are currently using this method to study both cell-type specific expression during development and stress, as well as heterogeneous cell populations at various stages of senescence.

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

A method for producing Arabidopsis leaf protoplasts that are compatible with fluorescence activated cell sorting (FACS), allowing for studies of specific cell populations. This method is compatible with any Arabidopsis line that expresses GFP in a subset of cells.

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

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

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)

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their prominent presence is often associated with poor prognosis1,2. CAFs are an activated subpopulation of stromal fibroblasts, many of which express the myofibroblast marker &alpha;-SMA3. CAFs originate from local tissue fibroblasts as well as from bone marrow-derived cells recruited into the developing tumor and adopt a CAF phenotype under the influence of the tumor microenvironment4. CAFs were shown to facilitate tumor initiation, growth and progression through signaling that promotes tumor cell proliferation, angiogenesis, and invasion5-8. We demonstrated that CAFs enhance tumor growth by mediating tumor-promoting inflammation, starting at the earliest pre-neoplastic stages9. Despite increasing evidence of the key role CAFs play in facilitating tumor growth, studying CAFs has been an on-going challenge due to the lack of CAF-specific markers and the vast heterogeneity of these cells, with many subtypes co-existing in the tumor microenvironment10. Moreover, studying fibroblasts in vitro is hindered by the fact that their gene expression profile is often altered in tissue culture11,12 . To address this problem and to allow unbiased gene expression profiling of fibroblasts from fresh mouse and human tissues, we developed a method based on previous protocols for Fluorescence-Activated Cell Sorting (FACS)13,14. Our approach relies on utilizing PDGFR&alpha; as a surface marker to isolate fibroblasts from fresh mouse and human tissue. PDGFR&alpha; is abundantly expressed by both normal fibroblasts and CAFs9,15 . This method allows isolation of pure populations of normal fibroblasts and CAFs, including, but not restricted to &alpha;-SMA+ activated myofibroblasts. Isolated fibroblasts can then be used for characterization and comparison of the evolution of gene expression that occurs in CAFs during tumorigenesis. Indeed, we and others reported expression profiling of fibroblasts isolated by cell sorting16. This protocol was successfully performed to isolate and profile highly enriched populations of fibroblasts from skin, mammary, pancreas and lung tissues. Moreover, our method also allows culturing of sorted cells, in order to perform functional experiments and to avoid contamination by tumor cells, which is often a big obstacle when trying to culture CAFs.

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

Cancer Associated Fibroblasts (CAFs) facilitate tumor initiation, growth and progression through signaling that promotes proliferation, angiogenesis, and inflammation. Here we describe a method to isolate pure populations of normal fibroblasts and CAFs from fresh mouse and human tissues by cell sorting, using PDGFR&#x03B1; as a surface marker.

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

Streptomycetes are filamentous soil bacteria that are used in industry for the production of enzymes and antibiotics. When grown in bioreactors, these organisms form networks of interconnected hyphae, known as pellets, which are heterogeneous in size. Here we describe a method to analyze and sort mycelial pellets using a Complex Object Parametric Analyzer and Sorter (COPAS). Detailed instructions are given for the use of the instrument and the basic statistical analysis of the data. We furthermore describe how pellets can be sorted according to user-defined settings, which enables downstream processing such as the analysis of the RNA or protein content. Using this methodology the mechanism underlying heterogeneous growth can be tackled. This will be instrumental for improving streptomycetes as a cell factory, considering the fact that productivity correlates with pellet size.

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

Liquid-grown Streptomyces cultures are characterized by mycelial pellets that are heterogeneous in size. We here describe a method to analyze and sort such pellets in a high-throughput manner. These pellets can be used for further analyses, which will provide leads to understand and control growth heterogeneity.

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

Endosomes are one of the major membrane sorting checkpoints in eukaryotic cells and they regulate recycling or destruction of proteins mostly from the plasma membrane and the Golgi. As a result the endosomal system plays a central role in maintaining cell homeostasis, and mutations in genes belonging to this network of organelles interconnected by vesicular transport, cause severe pathologies including cancer and neurobiological disorders. It is therefore of prime relevance to understand the mechanisms underlying the biogenesis and organization of the endosomal system. The yeast Saccharomyces cerevisiae has been pivotal in this task. To specifically label and analyze at the ultrastructural level the endosomal system of this model organism, we present here a detailed protocol for the positively charged nanogold uptake by spheroplasts followed by the visualization of these particles through a silver enhancement reaction. This method is also a valuable tool for the morphological examination of mutants with defects in endosomal trafficking. Moreover, it is not only applicable for ultrastructural examinations but it can also be combined with immunogold labelings for protein localization investigations.

Yeast, Saccharomyces cerevisiae, has been a key model organism to identify and study genes regulating the biogenesis and functions of the endosomal system. Here we present a detailed protocol for the specific labeling of the endosomal compartments for ultrastructural studies.

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

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific positions in DNA, RNA, proteins and small biomolecules. This natural methylation reaction can be expanded to a wide variety of alkylation reactions using synthetic cofactor analogues. Replacement of the reactive sulfonium center of AdoMet with an aziridine ring leads to cofactors which can be coupled with DNA by various DNA MTases. These aziridine cofactors can be equipped with reporter groups at different positions of the adenine moiety and used for Sequence-specific Methyltransferase-Induced Labeling of DNA (SMILing DNA). As a typical example we give a protocol for biotinylation of pBR322 plasmid DNA at the 5&#8217;-ATCGAT-3&#8217; sequence with the DNA MTase M.BseCI and the aziridine cofactor 6BAz in one step. Extension of the activated methyl group with unsaturated alkyl groups results in another class of AdoMet analogues which are used for methyltransferase-directed Transfer of Activated Groups (mTAG). Since the extended side chains are activated by the sulfonium center and the unsaturated bond, these cofactors are called double-activated AdoMet analogues. These analogues not only function as cofactors for DNA MTases, like the aziridine cofactors, but also for RNA, protein and small molecule MTases. They are typically used for enzymatic modification of MTase substrates with unique functional groups which are labeled with reporter groups in a second chemical step. This is exemplified in a protocol for fluorescence labeling of histone H3 protein. A small propargyl group is transferred from the cofactor analogue SeAdoYn to the protein by the histone H3 lysine 4 (H3K4) MTase Set7/9 followed by click labeling of the alkynylated histone H3 with TAMRA azide. MTase-mediated labeling with cofactor analogues is an enabling technology for many exciting applications including identification and functional study of MTase substrates as well as DNA genotyping and methylation detection.

DNA and proteins are sequence-specifically labeled with affinity or fluorescent reporter groups using DNA or protein methyltransferases and synthetic cofactor analogues. Depending on the cofactor specificity of the enzymes, aziridine or double activated cofactor analogues are employed for one- or two-step labeling.

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 ...