愿我们，感谢肯Nealson的整个手稿的准备过程中他的见解和建议。

1。病毒悬浮液的制备通过流式细胞仪分离出单个病毒颗粒之前，准备不固定的病毒悬液。 <ol><li>使用先前建立的协议，确保最终的病毒悬浮液中分离病毒颗粒被包含在0.1微米过滤TE液（Tris-EDTA，pH8）中。我们建议您使用其他方法虽然已开发出采用化学絮凝7切向流过滤（TFF）接近6。 </li><li>枚举使用既定的协议， 例如 ，使用萤光8-10或流式细胞仪11,12的病毒颗粒。 </li><li>病毒悬浮液的等分试样分成两个Eppendorf管中（最终体积是1,000微升）。 </li></ol> 2。流式细胞仪<ol><li>病毒使用BD FACSAria II流式细胞仪配备一个自定义的前向散射的PMT（FSC PMT）排序，稀释病毒颗粒在0.1微米过滤TE，pH值8。0到适当的事件发生率为200赛事秒-1滴度。 </li><li>设置阈值，以最大限度地提高信号与噪声的口粮， 例如 。含有T4和λ噬菌体颗粒的混合组合建议：在1000和SSC FSC PMT 200。 </li><li>运行100微升的“空白”样品只含有1）0.1微米过滤TE和2）0.1微米的过滤TE和SybrGreen1的1E-4的股票稀释（10,000倍）。 </li><li>运行一个小等分不染病毒悬浮液（100微升），以评估背景，其次是染病毒悬液（SybrGreen1 1E-4的股票稀释），病毒颗粒共5000事件。这是检查浓度和调整分配门，如有必要，在排序前。我们建议紧栅的中心的病毒颗粒。另外，门，我们使用的SYBR Green和FSC PMT情节。 </li><li>加入5μl的1％低熔点（LMP）琼脂糖（在TBE缓冲液）冷却至37℃到每个以及在一个聚四氟乙烯（PTFE）的显微镜载片上（电子显微学）。 </li><li>装入PTF​​E显微镜的幻灯片到流式细胞仪捕捉到排序的病毒颗粒。 </li><li> SYBR绿色染病毒颗粒直接到四氟滑板与LMP琼脂糖开始排序。我们建议使用10个或更多的事件（病毒颗粒）在某些井进行排序，以帮助在共聚焦激光扫描显微镜（CSLM）的深度的病毒的检测。 </li><li>当排序完成流式细胞仪，删除幻灯片，加入5微升LMP琼脂糖冷却到37°C，每孔，从而嵌入病毒颗粒（次）。 </li></ol> 3。可视化单的病毒粒子，利用共聚焦显微镜如果需要一个单一的病毒粒子，然后确定每个包含一个单独的病毒是势在必行，激光共聚焦显微镜是必要的，以可视化的嵌入式病毒颗粒（S）在3D验证，只有一个单一的粒子存在，而且多种病毒不堆叠在彼此的顶部。 <ol><li>置显微镜激光的波长488 nm激发。 </li><li>图片嵌入式病毒颗粒使用63X长工作距离的目标。 </li></ol> 4。全基因组扩增<ol><li>井与单一病毒一旦确定与激光共聚焦显微镜，琼脂糖（ 原位 ）内使用多重置换扩增（MDA）进行全基因组扩增。 </li><li>为了减少引入DNA污染的可能性，取出所需的琼脂糖珠从滑动，并放置在无菌的PCR管中。使用无菌镊子和/或刀片用于此步骤中的一对。 </li><li>裂解病毒粒子中就地采用热（94℃）的热循环仪的加热块中的3分。 </li><li>执行以下修改MDA反应下列制造商的建议（GenomiPhi HY试剂盒，GE医疗） <ol><li>减少体积的样品和反应BUF指11.25微升和孵化，在30°C，最多2小时，以减少非特异性扩增。 </li></ol></li><li>纯化扩增的基因组物质的基因组DNA（基因组DNA）转移到1.7毫升的Eppendorf管中，加1/10体积的3M醋酸钠（醋酸钠）在TBE缓冲液（LMP琼脂糖凝胶制剂中使用的相同的缓冲液）。 </li><li>将样品（次），在55℃10分钟以溶解琼脂糖。 </li><li>移动管（S）到42℃，以减少温度之前加入酶。 </li><li>新增2个β-琼胶每个管和孵化2小时。 </li><li>加入1倍体积的缓冲液饱和的苯酚溶液的试管中，剧烈混合，在3500×g离心15分钟，离心。将上清转移到新的1.7毫升的Eppendorf管中含有DNA。 </li><li>添加一个体积100％异丙醇和加入1μlGlycoblue。拌匀倒相管。 </li><li>在4℃以28,000 xg离心60分钟。倒出异丙醇，确保不打扰颗粒。加入150μl70％ETHA酚每个试管中。旋转28,000的XG及RT 10分钟。倒出乙醇，空气干燥DNA沉淀，并在适当体积的TE缓冲液中重悬DNA。 </li><li>我们建议以下修改重复步骤4.4-4.9。设置3-5复制MDA反应和减少孵化，在30℃至1小时。池样品后，纯化和沉淀物，用95％-100％的乙醇，以取得所需的浓度。 </li></ol> 5。代表结果 图1总结了SVG过程。这些方法使用流式细胞仪检测到病毒颗粒PTFE显微镜载玻片含琼脂糖分离单个的病毒颗粒从混合组合，然后由MDA获得数量足够进行测序的基因组DNA进行排序。 作为一个证明的概念，λ噬菌体T4混合SVG过程11。病毒颗粒一旦被抓获，并嵌入琼脂糖，激光共聚焦显微镜WA用来在不同可视化和确认一个单一的病毒粒子，得到的结果示于图2中，一旦与单个病毒粒子的孔中被确定，他们被选择为丙二醛（MDA）的基因组DNA。然后，我们使用的多重PCR具体T4和lambda来识别这种病毒的分离， 如图3所示。一个孤立的噬菌体基因组测序选择。 进行基因组测序，使用454-乙酸进行引用映射到识别的覆盖水平得到使用SVG的技术和测序读取。 如图4所示，几乎整个的λ基因组回收（与异常的第5个碱基）。 <img alt="图1" src="/files/ftp_upload/3899/3899fig1.jpg" /> 图1。 SVG方法。含有病毒悬液混合组合减少到单个病毒颗粒通过流式细胞仪则sorted到个人琼脂糖“珠”。该病毒嵌入在琼脂糖珠叠加一层额外的琼脂糖后流式细胞仪分选。最后， 在原位进行全基因组扩增。 <img alt="图2" src="/files/ftp_upload/3899/3899fig2.jpg" /> 图2。激光扫描共聚焦显微镜的排序病毒粒子嵌入在琼脂糖珠A）三维重建的SYBR Green I的的染病毒颗粒内的琼脂糖珠。插图：更高的放大倍率孤立的病毒粒子B）剖面图相对荧光染色的单个病毒颗粒内的琼脂糖珠。 <img alt="图3" src="/files/ftp_upload/3899/3899fig3.jpg" /> 图3。噬菌体鉴定采用PCR A）&lt;/ STRONG&gt;多重PCR使用T4和lambda特异性引物的基因型，车道：1。则TrackIT 1 kb的加入水口（Invitrogen公司），2。拉姆达整合（750 bp）的，3。 T4的主要衣壳蛋白（1050碱基对），4。混合的lambda整合和T4的主要衣壳蛋白B）后续的lambda与其他位点特异性PCR进一步证实噬菌体基因组隔离，车道：1。拉姆达整合（750 bp）的，2。拉姆达阻遏（356个基点）3。拉姆达SIE（二重感染排除）（456个基点）4。则TrackIT 1 KB加梯（Invitrogen公司）。 <img alt="图4" src="/files/ftp_upload/3899/3899fig4.jpg" /> 图4。拉姆达基因组属性和覆盖范围。）GC情节，B）的基因组图谱的lambda（改编自<a href="http://img.jgi.doe.gov" target="_blank">http://img.jgi.doe.gov</a> ），和C）的SVG读取映射参考λ噬菌体，x轴基因组的位置，雅红双喜％的覆盖率。

<ol>
	<li>Raghunathan, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+DNA+amplification+from+a+single+bacterium.">Genomic DNA amplification from a single bacterium.</a> Appl. Environ. Microbiol. 71, 3342-337 (2005).</li><li>Lasken, 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=Single-cell+genomic+sequencing+using+Multiple+Displacement+Amplification.">Single-cell genomic sequencing using Multiple Displacement Amplification.</a> Curr. Opin. Microbiol. 10, 510-516 (2007).</li><li>Ishoey, T., Woyke, T., Stepanauskas, R., Novotny, M., Lasken, 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=Genomic+sequencing+of+single+microbial+cells+from+environmental+samples.">Genomic sequencing of single microbial cells from environmental samples.</a> Curr. Opin. Microbiol. 11, 198-204 (2008).</li><li>Edwards, R. A., Rohwer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+metagenomics.">Viral metagenomics.</a> Nature Reviews Microbiology. 3, 504-510 (2005).</li><li>Rohwer, F., Prangishvili, D., Lindell, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+viruses+in+the+environment.">Roles of viruses in the environment.</a> Environ. Microbiol. 11, 2771-2774 (2009).</li><li>Williamson, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Sorcerer+II+Global+Ocean+Sampling+Expedition:+metagenomic+characterization+of+viruses+within+aquatic+microbial+samples.">The Sorcerer II Global Ocean Sampling Expedition: metagenomic characterization of viruses within aquatic microbial samples.</a> PLoS ONE. 3, e1456 (2008).</li><li>John, S. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+efficient+method+for+concentration+of+ocean+viruses+by+chemical+flocculation.">A simple and efficient method for concentration of ocean viruses by chemical flocculation.</a> Environ. Microbiol. Rep. 3, 195-202 (2011).</li><li>Noble, R., Fuhrman, J. <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+SYBR+Green+I+for+rapid+epifluorescence+counts+of+marine+viruses+and+bacteria.">Use of SYBR Green I for rapid epifluorescence counts of marine viruses and bacteria.</a> Aquat Microb Ecol. 14, 113-118 (1998).</li><li>Hara, S., Terauchi, K., Koike, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abundance+of+viruses+in+marine+waters:+assessment+by+epifluorescence+and+transmission+electron+microscopy.">Abundance of viruses in marine waters: assessment by epifluorescence and transmission electron microscopy.</a> Appl. Environ. Microbiol. 57, 2731-2734 (1991).</li><li>Hennes, K. P., Suttle, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+counts+of+viruses+in+natural+waters+and+laboratory+cultures+by+epifluorescence+microscopy.">Direct counts of viruses in natural waters and laboratory cultures by epifluorescence microscopy.</a> Limnol. Oceanogr. 40, 1050-1055 (1995).</li><li>Marie, D., Brussaard, C. P. D., Thyrhaug, R., Bratbak, G., Vaulot, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enumeration+of+marine+viruses+in+culture+and+natural+samples+by+flow+cytometry.">Enumeration of marine viruses in culture and natural samples by flow cytometry.</a> Appl. Environ. Microbiol. 65, (1999).</li><li>Brussaard, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enumeration+of+bacteriophages+using+flow+cytometry.">Enumeration of bacteriophages using flow cytometry.</a> Methods Mol. Biol. 501, 97-111 (2009).</li><li>Allen, L. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+Virus+Genomics:+A+New+Tool+for+Virus+Discovery.">Single Virus Genomics: A New Tool for Virus Discovery.</a> Plos One. 6, (2011).</li><li>Hutchison, C. A., Smith, H. O., Pfannkoch, C., Venter, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-free+cloning+using+phi+29+DNA+polymerase.">Cell-free cloning using phi 29 DNA polymerase.</a> Proceedings of the National Academy of Sciences of the United States of America. 102, 17332-17336 (2005).</li></ol>

没有利益冲突的声明。

应用SVG方法时，必须考虑一些重要因素。基因分型，在概念证明型实验13，是不是保守引物的环境或未知的菌株可在所有病毒组的有效选项。此外，背景DNA合成或非特异性扩增常见的扩增过程中使用MDA反应14。非特异性扩增已被归因于新兴的反应试剂和/或通过一种机制，允许从随机六聚体在反应混合物中的扩增的DNA污染。当使用病毒的组合，有可能是一个更高的可能性由于作为粒?...

<table border="1">
	<tbody>
		<tr>
			<td>Material Name</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>1X TE buffer</td>
			<td>Invitrogen</td>
			<td>12090-015</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Buffer-saturated Phenol</td>
			<td>Invitrogen</td>
			<td>15513-039</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>GenomiPhi HY kit</td>
			<td>GE Healthcare</td>
			<td>25-6600-22</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glycoblue</td>
			<td>Invitrogen</td>
			<td>AM9516</td>
			<td>15 mg/ml</td>
		</tr>
		<tr>
			<td>LMP agarose</td>
			<td>Invitrogen</td>
			<td>16520100</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PTFE microscope slide</td>
			<td>Electron Microscopy Sciences</td>
			<td>63430-04</td>
			<td>24 well, 4 mm Diameter</td>
		</tr>
		<tr>
			<td>SybrGreen</td>
			<td>Invitrogen</td>
			<td>S7585</td>
			<td>10,000X</td>
		</tr>
		<tr>
			<td>&beta;-agarase</td>
			<td>New England Biolabs</td>
			<td>M0392S</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

isolation and genome analysis of single virions using 'single virus genomics'

全基因组扩增和测序的单一微生物细胞使基因组特性，而不需要栽培1-3。病毒，是无处不在的，我们星球上的4最众多的实体和重要的在各种环境中5，通过类似的方法尚未透露。在这里，我们描述了一种方法，用于分离和表征单个病毒颗粒的基因组被称为“单个病毒的基因组学”（SVG）。 SVG利用流式细胞仪分离单个病毒和全基因组扩增，得到高分子量的基因组DNA（基因组DNA），可以用于在随后的测序反应。

Watch this Scientific Journal Video about Isolation and Genome Analysis of Single Virions using 'Single Virus Genomics' at JoVE.com

Isolation and Genome Analysis of Single Virions using &#039;Single Virus Genomics&#039;

单一的病毒基因组（SVG）是一种方法来隔离和放大的基因组单virons。病毒悬浮液的混合组合，用流式细胞仪检测到载玻片离散井含琼脂糖，从而捕获病毒粒子和减少下游加工过程中的基因组剪切排序。全基因组扩增是通过使用多重置换扩增（MDA）的产生，是适合用于测序的基因组物质中。

使用“单个病毒基因组的单病毒颗粒的分离和基因组分析&#39;

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which ...

isolation-genome-analysis-single-virions-using-single-virus

Research

JoVE Journal

Immunology and Infection

Isolation and Genome Analysis of Single Virions using 'Single Virus Genomics'

10.8K 次观看.  The J. Craig Venter Institute. 单一的病毒基因组（SVG）是一种方法来隔离和放大的基因组单virons。病毒悬浮液的混合组合，用流式细胞仪检测到载玻片离散井含琼脂糖，从而捕获病毒粒子和减少下游加工过程中的基因组剪切排序。全基因组扩增是通过使用多重置换扩增（MDA）的产生，是适合用于测序的基因组物质中。

视频: Isolation and Genome Analysis of Single Virions using 'Single Virus Genomics'

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in bioterrorism is also a concern. A better understanding of the cellular factors that impact infection would facilitate the development of much-needed therapeutics. Recent advances in RNA interference (RNAi) technology coupled with complete genome sequencing of several organisms has led to the optimization of genome-wide, cell-based loss-of-function screens. Drosophila cells are particularly amenable to genome-scale screens because of the ease and efficiency of RNAi in this system 1. Importantly, a wide variety of viruses can infect Drosophila cells, including a number of mammalian viruses of medical and agricultural importance 2,3,4. Previous RNAi screens in Drosophila have identified host factors that are required for various steps in virus infection including entry, translation and RNA replication 5. Moreover, many of the cellular factors required for viral replication in Drosophila cell culture are also limiting in human cells infected with these viruses 4,6,7,8, 9. Therefore, the identification of host factors co-opted during viral infection presents novel targets for antiviral therapeutics. Here we present a generalized protocol for a high-throughput RNAi screen to identify cellular factors involved in viral infection, using vaccinia virus as an example.

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Novel host factors involved in viral infection can be identified through cell-based genome-wide loss of function RNAi screening. A Drosophila cell culture model is particularly amenable to this approach due to the ease and efficiency of RNAi. Here we demonstrate this technique using vaccinia virus as an example.

RNAi筛选参与宿主因素牛痘病毒感染使用果蝇细胞

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in ...

科研

免疫与感染

Separation of Single-stranded DNA, Double-stranded DNA and RNA from an Environmental Viral Community Using Hydroxyapatite Chromatography

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2. Viruses modulate host cell abundance and diversity, contribute to the cycling of nutrients, alter host cell phenotype, and influence the evolution of both host cell and viral communities through the lateral transfer of genes 3. Numerous studies have highlighted the staggering genetic diversity of viruses and their functional potential in a variety of natural environments. 
Metagenomic techniques have been used to study the taxonomic diversity and functional potential of complex viral assemblages whose members contain single-stranded DNA (ssDNA), double-stranded DNA (dsDNA) and RNA genotypes 4-9. Current library construction protocols used to study environmental DNA-containing or RNA-containing viruses require an initial nuclease treatment in order to remove nontargeted templates 10. However, a comprehensive understanding of the collective gene complement of the virus community and virus diversity requires knowledge of all members regardless of genome composition. Fractionation of purified nucleic acid subtypes provides an effective mechanism by which to study viral assemblages without sacrificing a subset of the community&#x2019;s genetic signature. 
Hydroxyapatite, a crystalline form of calcium phosphate, has been employed in the separation of nucleic acids, as well as proteins and microbes, since the 1960s11. By exploiting the charge interaction between the positively-charged Ca2+ ions of the hydroxyapatite and the negatively charged phosphate backbone of the nucleic acid subtypes, it is possible to preferentially elute each nucleic acid subtype independent of the others. We recently employed this strategy to independently fractionate the genomes of ssDNA, dsDNA and RNA-containing viruses in preparation of DNA sequencing 12. Here, we present a method for the fractionation and recovery of ssDNA, dsDNA and RNA viral nucleic acids from mixed viral assemblages using hydroxyapatite chromotography.

We describe an efficient method to separate single-stranded DNA, double-stranded DNA and RNA molecules from environmental viral communities. Nucleic acids are fractionated using hydroxyapatite chromatography with increasing concentrations of phosphate-containing buffers. This method permits the isolation of all viral nucleic acid types from environmental samples.

分离单链DNA，双链DNA和RNA病毒使用羟基磷灰石色谱从环境社区

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2.  Viruses modulate host cell abundance and diversity, ...

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly constituted by proteins and exopolysaccharides, among other factors 7-10. The microbial community encased within the biofilm often shows the differentiation of distinct subpopulation of specialized cells 11-17. These subpopulations coexist and often show spatial and temporal organization within the biofilm 18-21.
Biofilm formation in the model organism Bacillus subtilis requires the differentiation of distinct subpopulations of specialized cells. Among them, the subpopulation of matrix producers, responsible to produce and secrete the extracellular matrix of the biofilm is essential for biofilm formation 11,19. Hence, differentiation of matrix producers is a hallmark of biofilm formation in B. subtilis.
We have used fluorescent reporters to visualize and quantify the subpopulation of matrix producers in biofilms of B. subtilis 15,19,22-24. Concretely, we have observed that the subpopulation of matrix producers differentiates in response to the presence of self-produced extracellular signal surfactin 25. Interestingly, surfactin is produced by a subpopulation of specialized cells different from the subpopulation of matrix producers 15.
We have detailed in this report the technical approach necessary to visualize and quantify the subpopulation of matrix producers and surfactin producers within the biofilms of B. subtilis. To do this, fluorescent reporters of genes required for matrix production and surfactin production are inserted into the chromosome of B. subtilis. Reporters are expressed only in a subpopulation of specialized cells. Then, the subpopulations can be monitored using fluorescence microscopy and flow cytometry (See Fig 1).
The fact that different subpopulations of specialized cells coexist within multicellular communities of bacteria gives us a different perspective about the regulation of gene expression in prokaryotes. This protocol addresses this phenomenon experimentally and it can be easily adapted to any other working model, to elucidate the molecular mechanisms underlying phenotypic heterogeneity within a microbial community.

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Microbial biofilms are generally constituted by distinct subpopulations of specialized cells. Single-cell analysis of these subpopulations requires the use of fluorescent reporters. Here we describe a protocol to visualize and monitor several subpopulationswithin B. subtilis biofilms using fluorescence microscopy and flow cytometry.

单细胞分析枯草芽孢杆菌生物膜，用荧光显微镜和流式细胞仪

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly ...

Visualizing Protein-DNA Interactions in Live Bacterial Cells Using Photoactivated Single-molecule Tracking

Protein-DNA interactions are at the heart of many fundamental cellular processes. For example, DNA replication, transcription, repair, and chromosome organization are governed by DNA-binding proteins that recognize specific DNA structures or sequences. In vitro experiments have helped to generate detailed models for the function of many types of DNA-binding proteins, yet, the exact mechanisms of these processes and their organization in the complex environment of the living cell remain far less understood. We recently introduced a method for quantifying DNA-repair activities in live Escherichia coli cells using Photoactivated Localization Microscopy (PALM) combined with single-molecule tracking. Our general approach identifies individual DNA-binding events by the change in the mobility of a single protein upon association with the chromosome. The fraction of bound molecules provides a direct quantitative measure for the protein activity and abundance of substrates or binding sites at the single-cell level. Here, we describe the concept of the method and demonstrate sample preparation, data acquisition, and data analysis procedures.

Photoactivated localization microscopy (PALM) combined with single-molecule tracking allows direct observation and quantification of protein-DNA interactions in live Escherichia coli cells.

可视化的活细菌细胞蛋白-DNA相互作用使用光活化的单分子跟踪

Protein-DNA interactions are at the heart of many fundamental cellular processes. For example, DNA replication, transcription, repair, and chromosome ...

Preparation of Single-cell Suspensions for Cytofluorimetric Analysis from Different Mouse Skin Regions

The skin is a barrier organ that interacts with the external environment. Being continuously exposed to potential microbial invasion, the dermis and epidermis home a variety of immune cells in both homeostatic and inflammatory conditions. Tools to obtain skin cell release for cytofluorimetric analyses are, therefore, very useful in order to study the complex network of immune cells residing in the skin and their response to microbial stimuli. Here, we describe an efficient methodology for the digestion of mouse skin to rapidly and efficiently obtain single-cell suspensions. This protocol allows maintenance of maximum cell viability without compromising surface antigen expression. We also describe how to take and digest skin samples from different anatomical locations, such as the ear, trunk, tail, and footpad. The obtained suspensions are then stained and analyzed by flow cytometry to discriminate between different leukocyte populations.

The skin is home to a complex immune cell network. We describe an efficient methodology for the digestion of mouse skin, from different parts of the animal's body, in order to obtain a single-cell suspension and analyze the different leukocyte populations resident in the skin by flow cytometry.

单细胞悬液的细胞荧光分析从不同的鼠标皮肤区域准备

The skin is a barrier organ that interacts with the external environment. Being continuously exposed to potential microbial invasion, the dermis and ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

中性粒细胞分离和分析，以确定他们的淋巴瘤细胞的敏感性对治疗药物的作用

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Highly Multiplexed, Super-resolution Imaging of T Cells Using madSTORM

Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and multiplexing ability. Using fluorescent nano-diamond fiducial markers, we describe the drift correction and alignment procedures required to obtain high precision in single molecule localization microscopy. In addition, a new multiplexing strategy, madSTORM, is described in which multiple molecules are targeted in the same cell using sequential binding and elution of fluorescent antibodies. madSTORM is demonstrated on an activated T cell to visualize the locations of different components within a membrane-bound, multi-protein structure called the T cell receptor microcluster. In addition, application of madSTORM as a general tool for visualization of multi-protein structures is discussed.

We demonstrate a method to image multiple molecules within heterogeneous nano-structures at single molecule accuracy using sequential binding and elution of fluorescently labeled antibodies.

使用madSTORM进行高分辨率，超分辨率的T细胞成像

Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and ...

Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies in an attempt to understand the precise mechanisms that lead to the breakdown of self-tolerance and subsequent autoimmunity. Approaches thus far have been based on the study of one specific aspect of the immune system (a single or few cell types or cytokines), and do not offer a global assessment of complex autoimmune disease. While patient sera-based studies have afforded important insights into autoimmunity, they do not provide the specific cellular source of the dysregulated cytokines detected. A comprehensive single-cell approach to evaluate cytokine production in multiple immune cell subsets, within the context of &#34;intrinsic&#34; patient-specific plasma circulating factors, is described here. This approach enables monitoring of the patient-specific immune phenotype (surface markers) and function (cytokines), either in its native &#34;intrinsic pathogenic&#34; disease state, or in the presence of therapeutic agents (in vivo or ex vivo).

Here we describe a single-cell proteomic approach to evaluate immune phenotypic and functional (intracellular cytokine induction) alterations in peripheral whole blood samples, analyzed via mass cytometry.

大鼠外周全血细胞免疫表型和因子生成的单细胞分析

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies ...

Isolation of Single Intracellular Bacterial Communities Generated from a Murine Model of Urinary Tract Infection for Downstream Single-cell Analysis

In this article, we outline a procedure used to isolate individual intracellular bacterial communities from a mouse that has been experimentally infected in the urinary tract. The protocol can be broadly divided into three sections: the infection, bladder epithelial cell harvesting, and mouth micropipetting to isolate individual infected epithelial cells. The isolated epithelial cell contains viable bacterial cells and is nearly free of contaminating extracellular bacteria, making it ideal for downstream single-cell analysis. The time taken from the start of infection to obtaining a single intracellular bacterial community is about 8 h. This protocol is inexpensive to deploy and uses widely available materials, and we anticipate that it can also be utilized in other infection models to isolate single infected cells from cell mixtures even if those infected cells are rare. However, due to a potential risk in mouth micropipetting, this procedure is not recommended for highly infectious agents.

This protocol describes a simple method of isolating single, infected-bladder epithelial cells from a murine model of urinary tract infection.

尿道感染小鼠模型产生的单细胞内细菌群落的分离, 用于下游单细胞分析

In this article, we outline a procedure used to isolate individual intracellular bacterial communities from a mouse that has been experimentally infected ...

Functionalization of Atomic Force Microscope Cantilevers with Single-T Cells or Single-Particle for Immunological Single-Cell Force Spectroscopy

Atomic force microscopy based single cell force spectroscopy (AFM-SCFS) is a powerful tool for studying biophysical properties of living cells. This technique allows for probing interaction strengths and dynamics on a live cell membrane, including those between cells, receptor and ligands, and alongside many other variations. It also works as a mechanism to deliver a physical or biochemical stimulus on single cells in a spatiotemporally controlled manner, thus allowing specific cell activation and subsequent cellular events to be monitored in real-time when combined with live-cell fluorescence imaging. The key step in those AFM-SCFS measurements is AFM-cantilever functionalization, or in other words, attaching a subject of interest to the cantilever. Here, we present methods to modify AFM cantilevers with a single T cell and a single polystyrene bead respectively for immunological studies. The former involves a biocompatible glue that couples single T cells to the tip of a flat cantilever in a solution, while the latter relies on an epoxy glue for single bead adhesion in the air environment. Two immunological applications associated with each cantilever modification are provided as well. The methods described here can be easily adapted to different cell types and solid particles.

We present a protocol to functionalize atomic force microscope (AFM) cantilevers with a single T cell and bead particle for immunological studies. Procedures to probe single-pair T cell-dendritic cell binding by AFM and to monitor the real-time cellular response of macrophages to a single solid particle by AFM with fluorescence imaging are shown.

原子力显微镜罐体功能化,具有单T细胞或单粒子免疫单细胞力光谱

Atomic force microscopy based single cell force spectroscopy (AFM-SCFS) is a powerful tool for studying biophysical properties of living cells. This ...