这项工作得到马克斯·普朗克社会的支持。

1.电生理的副溶血性弧菌细胞的制备和质粒DNA电泳到游泳细胞中注意:该协议的以下部分允许制备电感受态细胞，随后将质粒DNA电穿孔到游泳细胞中。如果荧光蛋白从质粒外显子表达，这个步骤很重要。 <ol><li>将来自-80℃甘油储备的V.副溶血弧菌细胞分离到含有100μg/ mL氨苄青霉素的新鲜LB琼脂平板上，并在37℃下孵育过夜。 </li><li>第二天，接种具有单个副溶血弧菌菌落的200mL LB培养基，并在37℃下在振荡条件下孵育，直到达到OD 600为1.0。孵化通常需要5-6小时才能使细胞培养达到所需的光密度。 </li><li>立即将细胞转移到冰上和水中在冰上和4°C预冷离心机中进行所有进一步的步骤。 </li><li>以2,000×g在4℃下收获细胞10分钟。 </li><li>通过倾析弃去上清液，并将细胞沉淀重新悬浮于25mL冰冷的273mM蔗糖溶液（pH7.4，用KOH缓冲）中。 </li><li>在2000×g下在4℃下再次收获细胞10分钟。重复两次。 </li><li>将洗涤的细胞沉淀物重新悬浮在400μL冰冷的273mM蔗糖溶液中。随后，加入甘油至最终浓度为15体积％体积。这些细胞现在可以进行电穿孔，但是也可以在-80℃的70μL等分试样中冷冻以供以后使用。 </li><li>对于电穿孔，将70μL等电量的电感受态细胞与100-1000ng质粒DNA混合，并将混合物转移到冰冷的电穿孔试管（0.2cm电极间隙）中。使用以下设置执行电穿孔:25μF，2400 V和200Ω。 </li><li>暂停在600μLLB肉汤中的电穿孔试管中的细胞，转移到2mL管中，并在37℃下振荡孵育3小时。 </li><li>将细胞以2,000 x g的速度旋转5分钟，并将沉淀重新悬浮在100μLLB肉汤中。将细胞悬浮液扩散到含有必需抗生素的选择性LB琼脂平板上，并在37℃下孵育过夜。 </li><li>第二天，检查盘子的集落形成。生长的菌落携带在质粒上编码的正确抗生素抗性基因，现在可用于进一步的实验。 </li></ol> 2.诱导Swarmer细胞分化注意:以下步骤描述了如何诱导副溶血弧菌分解为其更长的细胞生命周期以及如何刺激固体表面的增殖。 <ol><li>将4g心输液（HI）琼脂悬浮于100mL ddH 2 O中，以制备群组。 </li><li>小心煮沸HI琼脂溶液在微波炉中，不时摇动瓶子直到粉末溶解。在121℃高压灭菌20分钟。 </li><li>经高压灭菌后，将HI琼脂冷却至60-65℃。加入最终浓度为50μM的2,2&#39;-联吡啶（100％乙醇中50mM的储备溶液）。加入最终浓度为4mM的CaCl 2 （ddH 2 O中4M的储备溶液）。如有必要，在溶液中加入抗生素以维持质粒。 注意:如果计划使用诱导型质粒，请将诱导剂加入其最终浓度。 小心:2,2&#39;-联吡啶:急性毒性（口服，皮肤吸入）。使用手套和安全护目镜。 </li><li>将30 - 35 mL最终的HI琼脂溶液倒入150 mm培养皿中，让琼脂固化。 </li><li>在仔细观察细胞培养之前，将琼脂平板在37℃下干燥至少10分钟（或直到任何液体残留物都具有ppeared，但不再）右开一个敞开的盖子。 注意:干燥时间高度依赖于使用的培养箱，严重干燥的板不会允许副溶血弧菌的聚集 。 （这一步非常重要！）板必须使用新鲜，不应超过2 - 3小时。旧板太干了，不能刺激群众。 </li><li>将少量细胞从单个菌落的边缘接种到5mL LB肉汤中。孵育培养物在37℃振荡直至达到OD 600 = 0.8，通常需要2-3小时。之后，细胞准备好被发现到HI群体琼脂平板上。 </li><li>将1μL细胞培养物置于HI琼脂积聚板的中心（ 图1A ）。 </li><li>让斑点干燥（ 图1B ）。 </li><li>用透明胶带密封板，并确保其牢固连接，否则可能会在过夜孵育期间分离（ 图1C ）。 注意:非常重要的是，密封是完美的。应用时，避免气泡的形成和胶带折叠。 </li><li>将板在24℃孵育过夜。这将导致从群集周边延伸的群聚体形成副溶血弧菌群落（ 图2 ）。 </li></ol>荧光显微镜检查副溶血弧菌Swarmer细胞的制备注意:以下协议描述了如何制备显微镜琼脂糖载玻片和随后将琼脂糖斑块印刷到琼脂糖垫上，以获得单一的微泡显微镜细胞。 <ol><li>为了制备用于显微镜的琼脂糖载玻片，将1g琼脂糖加入到100mL的20体积/体积的PBS和10％vol / vol的LB肉汤溶液中。小心地煮沸微波溶液溶解琼脂糖，让其冷却至65-70℃，同时用磁力搅拌器混合。 </li><li>放置ac瘦肉显微镜滑到工作台上的完美水平部分。用两层通用实验室标签胶带盖住滑块的两端，将滑块固定到工作台上。两条胶带之间的距离应为3厘米左右。 注意:两层胶带产生非常小的高度，这将确定琼脂糖垫的高度。重要的是，工作空间是水平的，以确保细胞将在同一平面进行显微镜分析。 </li><li>将Pipet〜250μL先前制备的琼脂糖溶液加到非覆盖的玻璃表面上。 </li><li>现在放置第二个显微镜载玻片与顶部底部相同的方向，稍微按下它，使残留的琼脂糖可以流出到两侧。 </li><li>让琼脂糖固化1-2分钟，然后慢慢地将顶部的玻璃滑板从底部的水平运动中拉出。 </li><li>使用手术刀，切断所有残留的琼脂糖显微镜的两侧滑动并从显微镜载玻片的末端缓慢取下胶带。 注意:琼脂糖载玻片现在已经准备就绪，应尽快使用，因为它会慢慢开始变干。 </li><li>为了在swellmer细胞上进行显微镜检查，从群集群体的最外边缘切出约3毫米×10毫米的琼脂琼脂（ 图1D ）。直到将swellmer细胞转移到显微镜载玻片之前，不要破坏群盘的胶带密封是非常重要的。一旦去除了胶带， 聚集的副溶血性细胞就会分化为游泳细胞。 注意:确保将切片从群集边缘外侧引导到菌落内部（ 图1D ）。这样就可以防止"游泳者污染"进入群众的边缘。 </li><li>将琼脂糖片转移到显微镜琼脂糖载玻片上，细胞朝向琼脂糖片d（ 图1E ）。这样将这些细胞印迹到琼脂糖垫上。 </li><li>等待〜30秒后，从琼脂糖垫中小心地取出琼脂片（ 图1F ）。 </li><li>将玻璃盖玻片放在琼脂糖垫上，细胞被印记。这些细胞现在可以用于显微镜。 注意:重要的是立即对swellmer细胞进行成像，因为swellmer细胞缓慢地向游泳者状态分化。 </li></ol> 4.副溶血素游离细胞培养物的制备用于荧光显微镜注意:该方案的这一部分描述了如何准备副溶血弧菌的培养物，以对游泳细胞进行荧光显微镜观察。 <ol><li>将少量细胞从单个菌落的边缘接种到5mL LB肉汤中。孵育培养物在37℃振荡1小时或直到轻微的细胞生长变得可见。 </li><li>在如果需要，该阶段添加用于表达目的蛋白质的诱导剂。在该实施例中，为了诱导YFP-CheW9的表达，加入L-阿拉伯糖至终浓度为0.2％（w / vol）。 </li><li>将培养物在37℃下再培养2小时。 </li><li>按照步骤3.1-3.7准备显微镜琼脂糖载玻片。 </li><li>在琼脂糖垫的中心点点1μL细胞培养物，让斑点孵育直至干燥。 </li><li>将玻璃盖玻片放在琼脂糖垫上，细胞被发现。现在准备在荧光显微镜下成像细胞。 </li></ol>图像分析该协议的这一部分描述了图像分析的工作流程，特别是如何提取用于人口统计学分析的单细胞荧光数据。 <ol><li>在开始图像分析之前，请确保所有显微镜图像都以16位TIF格式保存。 </li><li>将DIC（或相位对比度）和相应的荧光通道图像加载到软件中。 </li><li>通过选择"显示/覆盖图像"生成两个通道的叠加图像。将图像数（"＃图像:"）设置为2，并选择第一个通道中的DIC图像和第二个通道中的荧光图像。 </li><li>从工具栏中选择"多行"工具，将单元格从一个极点标记到另一个单元格的中间。为了获取所有生成的行（行对象）的列表打开"测量/区域测量"。配置区域测量仅显示区域标签，距离和平均强度。 </li><li>将"测量/校准距离"下的像素大小校准为显微镜设置特定的像素大小，然后单击"应用于所有打开的图像"。 </li><li>将所有区域（线对象）转移到荧光通道图像。在"地区/运输"er Regions"选择源图像（叠加图像）和目的图像（荧光通道图像），选择"所有区域"，然后按"确定"。 注意:为了能够以后重新生成精确的行（行对象，区域，ROI），请打开"区域/保存区域"并保存区域。 </li><li>通过打开"测量/区域测量"并按"打开日志"将所有区域测量结果导出到电子表格中。确认打开并导出数据。一旦电子表格在后台打开，最后按"F9:Log Data"导出数据。 </li><li> 要确定荧光灯沿着细胞的位置，打开"测量/线条扫描"。将"Linescan宽度"设置为一个值，以便单元格的整个宽度被先前创建的区域覆盖。右键单击行扫描图，然后选择"显示图形数据"。通过左键单击并将鼠标拖放到行扫描中兴趣点，"图形数据"窗口将显示相应的像素/两点之间的距离。选择蓝色突出显示的行，然后复制内容。 注意:通过在"Linescan"窗口中按"日志数据"，可以将整个行扫描强度配置文件记录到电子表格中。提取的数据现在可以用于图形中的可视化或进一步的统计分析。在细胞周期的细胞长度和细胞体的荧光强度的人口统计学表示中，可以容易地观察细胞周期上的定位模式。这些表示可以使用先前选择的相同ROI获得。以下步骤将给出如何生成类似于图3C和4C所示的人口统计的示例。 <ol><li>在先前在软件中创建的Fiji / ImageJ加载区域标记（.rgn）（步骤5.6）中使用"Metamorph nd＆ROI files importer";。或者，可以使用内置的"分段线"工具在Fiji / ImageJ中直接创建要分析的单元的感兴趣区域（ROI）。 </li><li>打开"分析/工具/投资回报率管理器"并标记所有活动列表条目"。 </li><li>之后，点击"更多/多图"，然后在新的"多图"窗口中选择"列表"。点击一个表项，然后选择并"全部复制"。随后，粘贴到一个新的电子表格。 注意:请确保电子表格中最长单元格的数据（两行对应的X和Y行数最多的行）是最右边的数据。 </li><li>将电子表格另存为"CSV（逗号分隔）（* .csv）"。 </li><li>从这里下载R脚本"https://github.com/ta-cameron/Cell-Profiles"10。脚本按照长度排序细胞，并将所有细胞的荧光强度分布归一化为每个细胞荧光素的平均值即</li><li>在R [版本3.0.1中运行整个脚本"单元格配置文件function.R" 11 ]。 </li><li>编辑第30行中的"example plots.R"脚本，以便文件路径引用保存以前生成的.csv文件的文件夹。另外，将第31行中的文件名"profile.csv"更改为在步骤5.9.4中生成的.csv文件所选择的文件名。然后，运行"example plots.R"，并按照"https://github.com/ta-cameron/Cell-Profiles"中的文档以及脚本文件中的注释生成人口统计分析。 </li></ol></li></ol>

<ol>
	<li>McCarter, L., Hilmen, M., Silverman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flagellar+dynamometer+controls+swarmer+cell+differentiation+of+V.+parahaemolyticus.">Flagellar dynamometer controls swarmer cell differentiation of V. parahaemolyticus.</a> Cell. 54 (3), 345-351 (1988).</li><li>Kawagishi, I., Imagawa, M., Imae, Y., McCarter, L., Homma, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+sodium-driven+polar+flagellar+motor+of+marine+Vibrio+as+the+mechanosensor+that+regulates+lateral+flagellar+expression.">The sodium-driven polar flagellar motor of marine Vibrio as the mechanosensor that regulates lateral flagellar expression.</a> Mol. Microbiol. 20 (4), 693-699 (1996).</li><li>Ulitzur, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+swarming+in+Vibrio+parahaemolyticus.">Induction of swarming in Vibrio parahaemolyticus.</a> Arch. Microbiol. 101 (4), 357-363 (1974).</li><li>Ulitzur, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+temperature,+salts,+pH,+and+other+factors+on+the+development+of+peritrichous+flagella+in+Vibrio+alginolyticus.">Effect of temperature, salts, pH, and other factors on the development of peritrichous flagella in Vibrio alginolyticus.</a> Arch. Microbiol. 104 (3), 285-288 (1975).</li><li>Ulitzur, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanism+of+swarming+of+Vibrio+alginolyticus.">The mechanism of swarming of Vibrio alginolyticus.</a> Arch. Microbiol. 104 (1), 67-71 (1975).</li><li>Gode-Potratz, C. J., Chodur, D. M., McCarter, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+and+iron+regulate+swarming+and+type+III+secretion+in+Vibrio+parahaemolyticus.">Calcium and iron regulate swarming and type III secretion in Vibrio parahaemolyticus.</a> J. Bacteriol. 192 (22), 6025-6038 (2010).</li><li>McCarter, L., Silverman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron+regulation+of+swarmer+cell+differentiation+of+Vibrio+parahaemolyticus.">Iron regulation of swarmer cell differentiation of Vibrio parahaemolyticus.</a> J. Bacteriol. 171 (2), 731-736 (1989).</li><li>Gode-Potratz, C. J., Kustusch, R. J., Breheny, P. J., Weiss, D. S., McCarter, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+sensing+in+Vibrio+parahaemolyticus+triggers+a+programme+of+gene+expression+that+promotes+colonization+and+virulence.">Surface sensing in Vibrio parahaemolyticus triggers a programme of gene expression that promotes colonization and virulence.</a> Mol. Microbiol. 79 (1), 240-263 (2011).</li><li>Heering, J., Ringgaard, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+Localization+of+Chemotactic+Signaling+Arrays+during+the+Lifecycle+of+Vibrio+parahaemolyticus.">Differential Localization of Chemotactic Signaling Arrays during the Lifecycle of Vibrio parahaemolyticus.</a> Front Microbiol. 7 (November), 1-12 (2016).</li><li>Cameron, T. A., Anderson-Furgeson, J., Zupan, J. R., Zik, J. J., Zambryski, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptidoglycan+synthesis+machinery+in+Agrobacterium+tumefaciens+during+unipolar+growth+and+cell+division.">Peptidoglycan synthesis machinery in Agrobacterium tumefaciens during unipolar growth and cell division.</a> mBio. 5 (3), (2014).</li><li>Development Core Team, R. F. F. S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=R:+A+Language+and+Environment+for+Statistical+Computing.">R: A Language and Environment for Statistical Computing.</a> R Foundation for Statistical Computing. 1, 2673 (2008).</li><li>Ringgaard, S., 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=ParP+prevents+dissociation+of+CheA+from+chemotactic+signaling+arrays+and+tethers+them+to+a+polar+anchor.">ParP prevents dissociation of CheA from chemotactic signaling arrays and tethers them to a polar anchor.</a> Proc Natl Acad Sci U S A. 111 (2), E255-E264 (2014).</li><li>Ringgaard, S., Schirner, K., Davis, B. M., Waldor, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+family+of+ParA-like+ATPases+promotes+cell+pole+maturation+by+facilitating+polar+localization+of+chemotaxis+proteins.">A family of ParA-like ATPases promotes cell pole maturation by facilitating polar localization of chemotaxis proteins.</a> Genes Dev. 25, 1544-1555 (2011).</li><li>Vischer, N. O. E., 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=Cell+age+dependent+concentration+of+Escherichia+coli+divisome+proteins+analyzed+with+ImageJ+and+ObjectJ.">Cell age dependent concentration of Escherichia coli divisome proteins analyzed with ImageJ and ObjectJ.</a> Front Microbiol. 6 (JUN), (2015).</li><li>Schneider, C. a., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nat. Methods. 9 (7), 671-675 (2012).</li><li>Sliusarenko, O., Heinritz, J., Emonet, T., Jacobs-Wagner, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput,+subpixel+precision+analysis+of+bacterial+morphogenesis+and+intracellular+spatio-temporal+dynamics.">High-throughput, subpixel precision analysis of bacterial morphogenesis and intracellular spatio-temporal dynamics.</a> Mol. Microbiol. 80 (3), 612-627 (2011).</li><li>Ducret, A., Quardokus, E. M., Brun, Y. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicrobeJ,+a+tool+for+high+throughput+bacterial+cell+detection+and+quantitative+analysis.">MicrobeJ, a tool for high throughput bacterial cell detection and quantitative analysis.</a> Nat. Microbiol. 1, 16077 (2016).</li><li>Carpenter, A. E., 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=CellProfiler:+image+analysis+software+for+identifying+and+quantifying+cell+phenotypes.">CellProfiler: image analysis software for identifying and quantifying cell phenotypes.</a> Genome Biol. 7 (10), R100 (2006).</li><li>Morales-Soto, N., 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=Preparation,+Imaging,+and+Quantification+of+Bacterial+Surface+Motility+Assays.">Preparation, Imaging, and Quantification of Bacterial Surface Motility Assays.</a> J. Vis. Exp. (98), e52338 (2015).</li><li>Paintdakhi, 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=Oufti:+An+integrated+software+package+for+high-accuracy,+high-throughput+quantitative+microscopy+analysis.">Oufti: An integrated software package for high-accuracy, high-throughput quantitative microscopy analysis.</a> Mol. Microbiol. 99 (4), 767-777 (2016).</li><li>de Jong, I. G., Beilharz, K., Kuipers, O. P., Veening, J. -. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+Cell+Imaging+of+Bacillus+subtilis+and+Streptococcus+pneumoniae+using+Automated+Time-lapse+Microscopy.">Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy.</a> J. Vis. Exp. (53), e1-e6 (2011).</li></ol>

没有利益冲突声明。

本文报告了一种用于副溶血性弧菌细胞的显微镜成像的方法，随后进行下游分析，旨在解决和量化研究的荧光标记蛋白质的亚细胞定位和其他特征。虽然对于显微镜图像处理和分析存在几种工具14,15,16,17,18，副溶血弧菌聚集细胞的分析仍然是具有挑战性的，因为它们通常存在于密集的细胞群中，并且细胞长度的变化很大（ 图2 ）。因此，自动细胞检测有时是错误的，因此结果有些不可靠。该协议概述了一条管道，旨在增长，形象和分析副溶血弧菌的群体细胞，这是根据规范量身定制的这些细胞的需要，例如表面上的受控生长环境和下游图像分析，其适应在接近生长的可变长度的细胞的存在。 从质粒外显子表达感兴趣的蛋白质的能力对于几个实验是重要的， 例如荧光标记的蛋白质的表达用于显微镜研究或基因缺失的互补。这里还描述了制备化学感受态细胞和将质粒DNA 电穿孔到副溶血弧菌中的过程。通常用于副溶血弧菌的电穿孔对缀合的优点是，质粒骨架不需要被修饰以允许缀合。不过，应采取一些预防措施;如步骤1.2所述，达到OD 600 = 1.0的细胞密度是非常重要的。在较低的光密度下，细胞会在电解后溶解在高密度下，质粒不被细胞吸收。此外，副溶血性弧菌细胞的离心太苛刻（步骤1.4-1.6）会大大降低电穿孔效率。在洗涤步骤期间仔细重新悬浮细胞沉淀物以产生电感受态细胞也是重要的。 虽然细胞培养的准备是微不足道的，文化密度对于群体性能来说非常重要。通常，早期指数生长期（OD 600 &lt;0.5）的细胞培养物在夜间孵育期间将比晚期指数或早期固定期的培养物产生更大的群集。根据实验，较大的群集可能是有益的。然而，为了比较在相同的HI琼脂块群上生长的不同菌株之间的菌落扩增和形态，重要的是在个体群集之间留有足够的空间。这可以是ach即通过发现较高光密度的细胞培养物，这通常会导致较小的群集。 诱导的群体表型在细菌种类中有显着差异，其重现性受到琼脂和营养成分，环境温度和周围环境和生长培养基的水分含量等因素的高度影响。因此，方案和外部环境的微小变化显着影响群体测定结果和诱导快速分化。已经报道了用于诱导其它细菌种群如铜绿假单胞菌和枯草芽孢杆菌 群的方案 。本协议中描述的步骤将可靠地产生更高细胞型的副溶血弧菌 ，并将其维持在群集边缘产生单层群聚火炬的群集群的形式。执行稳定和对副溶血性弧菌细胞类型的重复实验 ，HI群体琼脂平板的制备是该方案中最重要的部分。特别地，干燥步骤可能需要优化，因为每个孵化器将表现不同。影响群众行为的另一个因素是培养皿中使用的塑料类型。我们实验室使用的典型培养皿进行群聚实验由聚苯乙烯（PS）制成。然而，使用由低密度聚乙烯（PE LD）制成的培养皿在群体测定中产生混合结果。遵循所描述的步骤是至关重​​要的，因为仅形成显示不同周边光斑形成的可重现的群集，这使得能够进行单次Swarmer细胞显微术。 毫无疑问，可以采用不同的方法来显示通过荧光显微镜研究副溶血弧菌聚集细胞的结果。对于instance，广泛使用的程序MicrobeTracker 16代表了一个合理的替代方案。最近可用的MicrobeTracker应用程序Oufti 20 （不需要MATLAB）也是另一种选择。然而，在Oufti中使用差分界面对比（DIC）图像是不可能的，相位对比成像不适用于在swarmer细胞中辨别细胞分割。此外，不是所有的显微镜都配备了相位对比成像，反过来相位对比成像也不适用于各种显微镜分析。另一个有前景的工具是免费的，基于ImageJ的是MicrobeJ 17 ，但是再次只能使用相位对比度和荧光图像。此外，该协议仅允许在其生命周期的特定时间点对细胞进行成像和分析。然而，描述延时成像和随后的图像分析的优秀协议允许以下单个细胞轨迹和蛋白质动力学21也可能应用于副溶血弧菌 。 按照本协议中描述的步骤，科学家们可以在游泳者和更高细胞型的副溶血弧菌上进行可靠和可再生的单细胞荧光显微镜检查 。该协议详细介绍了将副溶血弧菌从其游泳者转化为其更细胞型的可靠方法。通过将来自群集群的外周斑马的单层的单层印迹到琼脂糖垫上，可以观察到蛋白质的细胞内定位，用于图像分析的管道可能适用于细菌生长在细菌群落内的其他细菌种类非常接近的地方和自动图像分析可能不是有利的情况。

细菌不断经历对外部环境的变化，并开发了几种技术来相应地改变和调整其行为。一种这样的机制涉及分化为更好地补充改变的环境的不同细胞类型。分化通常涉及细胞周期调控，细胞形态和细胞时空组织的重大变化。可以进行分化的一种生物体是副溶血弧菌（Vibrio parahaemolyticus） 。 副溶血性弧菌属于Vibrionaceae ，它是通常居住在新鲜或咸水中的一类蛋白菌。 伏立木科植物广泛分布在环境中，包括引起人类肠道感染的若干物种，也包括霍乱弧菌。副溶血弧菌是一个二态生物，能够分化成两种不同的细胞类型，作为对其变化的反应外部环境在水环境中，它作为一个短杆形游泳细胞存在，单个极鞭毛位于老细胞柱上。在表面接触时，触发分解成更细胞的细胞。 Swarmer细胞分化涉及两个主要变化:通过抑制细胞分裂使细胞形态发生变化，并诱导第二鞭毛系统。这导致形成了围绕和高度细长的棒状的旋转细胞，其可以继续使用更生命的生活方式，其中分裂事件导致后代的swarmer细胞，或者分化回游泳细胞。 据报道有几个因素诱导或影响sw mer分化。主要刺激似乎是通过机械感应驱动的，其中副溶血性弧菌使用极鞭毛作为触觉传感器，其检测表面接触时的旋转抑制，但是涉及其他几个因素好1 。在实验室中，极地鞭毛的旋转可以通过加入苯胺进行人为的抑制，其阻止钠通道驱动的鞭毛旋转，从而诱导更多的分化2 。此外，在早期的研究中，当细胞在用透明塑料胶带密封的培养皿中的生长培养基上繁殖时，诱导溶解在溶解固体培养基上的溶弧弧菌 。碱饱和过滤纸在这些条件下阻止了群集，因此表明一个或多个挥发性酸可能参与诱导群集。因此，用塑料胶带密封培养皿可能允许在板的顶部空间内积聚作为细胞新陈代谢的副产物的挥发性酸。当在未密封的培养皿中将H 2 O 2加入到生长培养基中以通过水解培养基成分人工产生挥发性酸时，获得了相同的效果<supclass ="xref"&gt; 3，4，5 。然而，这种挥发性酸的身份仍然是未知的。此外，已经表明，钙6和铁限制7的过量可用性都增强了在固体表面上的分散和增殖。通过将化合物2,2&#39;-联吡啶加入到生长培养基中，可以使细胞饥饿，这已经显示出影响swarmer分化8 。已知调节分化的因素现在在可重复地诱导副溶血弧菌分化成swarmer细胞的方案的设计中实现，并且在固体琼脂表面上增殖。 副溶血性分化涉及细胞分裂调控，细胞形态学，大分子机器如flagell的定位的重大变化a和趋化性装置 - 根据细胞周期都需要蛋白质的特异性定位的过程。因此，研究这些蛋白质的细胞内定位的能力对于理解上述细胞过程是至关重要的。为了进行这样的研究，需要在单细胞上进行荧光显微镜检查。当在密集细菌群体中存在的细胞成像时，这可能是特别有挑战性的，如同泡沫细胞的情况。已经尝试在液体培养基中诱导更多的分化，这可能潜在地产生用于显微镜研究的单细胞。尽管在转录水平上，这些细胞已经部分地诱导了Swarmer分化程序，但是它们不像在固体培养基8上生长的完全分化的swarmer细胞经历相同的形态学变化。本文提供了一种强大且可重复的方法来诱导swarmer ce的方法在琼脂表面上分化。该方案产生容易获得的swarmer细胞群，可用于单细胞显微镜和随后的分析。此外，该方案可以使游泳者和swarmer细胞类型中的荧光标记蛋白质的定位研究（部分1,2,3,4）。此外，该协议描述了如何处理和分析来自荧光显微镜实验的生成数据的后续工作流程，其允许细菌学社会的人口统计学分析（第5节）。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Media components</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LB-medium</td>
			<td>Roth</td>
			<td>X968.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Difco Agar, granulated</td>
			<td>BD</td>
			<td>214510</td>
			<td>For preparation of LB agar with LB-medium</td>
		</tr>
		<tr>
			<td>Difco Heart Infusion Agar</td>
			<td>BD</td>
			<td>244400</td>
			<td>For preparation of HI agar swarming plates</td>
		</tr>
		<tr>
			<td>Agarose NEEO, ultra quality</td>
			<td>Roth</td>
			<td>2267.3</td>
			<td>For preparation of microscopy agarose pads</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Additives</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>L-arabinose</td>
			<td>Roth</td>
			<td>5118.3</td>
			<td>Used to induce pBAD derivatives</td>
		</tr>
		<tr>
			<td>2,2`-Bipyridyl</td>
			<td>Sigma Aldrich</td>
			<td>D216305-25G</td>
			<td>For preparation of HI agar swarming plates</td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate</td>
			<td>Roth</td>
			<td>5239.1</td>
			<td>For preparation of HI agar swarming plates</td>
		</tr>
		<tr>
			<td>Ampicillin sodium salt</td>
			<td>Roth</td>
			<td>K029.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloramphenicol</td>
			<td>Roth</td>
			<td>3886.3</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS buffer</td>
			<td>-</td>
			<td>-</td>
			<td>Standard recipe for Phosphate-buffered saline</td>
		</tr>
		<tr>
			<td>D(+) Sucrose</td>
			<td>Roth</td>
			<td>4621.1</td>
			<td>For preparation of electrocompetent cells</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Roth</td>
			<td>3783.5</td>
			<td>For preparation of electrocompetent cells</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Hardware</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish 150x20mm with cams</td>
			<td>Sarstedt</td>
			<td>82.1184.500</td>
			<td>For preparation of HI agar swarming plates</td>
		</tr>
		<tr>
			<td>kelvitron t (B 6420)</td>
			<td>Heraeus</td>
			<td>-</td>
			<td>Used for drying HI agar swarming plates</td>
		</tr>
		<tr>
			<td>Gene Pulser Xcell Microbial System</td>
			<td>BioRad</td>
			<td>1652662</td>
			<td>Used for elctroporation of plasmid DNA</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MetaMorph Offline (version 7.7.5.0)</td>
			<td>Molecular Devices</td>
			<td>-</td>
			<td>Used for microscopy image analysis</td>
		</tr>
		<tr>
			<td>Excel</td>
			<td>Microsoft</td>
			<td>-</td>
			<td>Used for microscopy data analsis</td>
		</tr>
	</tbody>
</table>

induction of cellular differentiation and single cell imaging of vibrio parahaemolyticus swimmer and swarmer cells

研究蛋白质的细胞内定位的能力对于了解许多细胞过程是至关重要的。反过来，这需要获得用于荧光显微镜的单细胞的能力，当在细菌群落内存在细胞时，这可能是特别具有挑战性的。例如，人类病原体副溶血弧菌在液体条件下作为短棒状游泳细胞存在，其在表面接触时分化成专门用于在固体表面上生长的高度细长的漂浮细胞的亚群。本文提出了一种在其两种差异状态下进行副溶血弧菌的单细胞荧光显微镜分析的方法。该协议非常可重复地诱导副溶血弧菌分解成更细胞的生命周期，并促进其在固体表面上的增殖。该方法产生从t延伸的分化的swarmer细胞的耀斑他的群体边缘。值得注意的是，在群星的尖端，细胞细胞存在于单层细胞中，这允许它们容易地转移到显微镜载玻片上并随后进行单细胞的荧光显微镜成像。此外，介绍了细菌社会人口统计学图像分析工作流程。作为原理证明，描述了溶血性弧菌的游泳者和swarmer细胞中趋化性信号阵列的细胞内定位的分析。

诱导分化和产生群集菌落 图1提供了生产副溶血弧菌群落的重要步骤的模式（ 图1A-C ）。游泳者培养物被发现在群体琼脂上，并在24 ℃下孵育，在固体琼脂表面上诱发更多的分化和增殖。 图2中给出了群集群体的代表性立体显微镜图像。立体显微镜成像清楚地显示了从群集周边向外延伸的群体。较高的放大倍数显示，在群体中，细胞仅分成单层或双层，而在群集细胞的中间，细胞堆叠成多层（ 图2A ）。群发喇叭可以很容易地转移（如步骤3.8-3.11中所述， <strong&gt;图1）到显微镜载玻片。重要的是，耀斑仅由完全分化的泡沫细胞群组成（ 图2B ）。这与群集中间的细胞相比，这些细胞明显更短，可能代表游泳者和泡沫细胞的混合物（ 图2B ）。此外，将耀斑小心地转移到显微镜载玻片上，使群体的整体结构和群体的边缘完整无缺，导致单层细胞可以单细胞显微镜访问。当在显微镜载玻片上印刷群发荧光时，尽可能平静地进行转印是非常重要的，而不是增加太多的压力 - 否则，来自群星光斑的细胞将会散布在与非起泡细胞在群集的中间。 荧光显微镜和图像分析<pclass ="jove_content"fo:keep-together.within-page ="1"&gt;作为差异不同的副溶血性游泳细胞和swarmer细胞的单细胞显微镜原理的证明，进行了异位表达的YFP-CheW的详细分析。 CheW是必需的趋化性蛋白，可用作趋化性信号转导阵列9,12的细胞内定位的标志物。在L-阿拉伯糖诱导型启动子下编码YFP-CheW的质粒被电穿孔到副溶血性弧菌中 ，如方案的第1节所述。对于显微镜，分别如第4节和第3节所述制备游泳者和泡沫细胞。如前所述，将YFP-CheW定位在单极性（ 图3A ，红色箭头）和双极性方式（ 图3A ，黄色箭头） 12,13 。因此，鉴定细胞极a在不同长度的YFP-蛋白质定位的变化提供了如何随着细胞周期的进展定位模式演变的很好的见解。然后可以将分散图中的变量"单元长度"和"焦点到单元极的距离"相关联的这些数据显示（图3B）。为了避免使用自动细胞检测工具时常见的轮廓检测中的不精确，通过使用MetaMorph中的"多行"选项或"分段行"中的"多行"选项，通过手动将线从一个极点绘制到另一个极点来选择单元格开源平台ImageJ。随后，可以收集所选择的感兴趣区域（ROI）的强度分布，允许识别YFP-蛋白质显示其最高强度的点以及细胞的总长度。如第5节所述分析单细胞的荧光谱，并通过绘制焦点与细胞极的距离来呈现作为细胞长度的函数（ 图3B ）。该分析清楚地表明，短细胞仅显示YFP-CheW的单极定位模式，而在较长的细胞中，YFP-CheW是双极定位的。此外，细胞周期中的定位模式可以容易地以细胞长度和细胞体的荧光强度的人口统计学表示来显现。这些表示可以使用在准备散点图时先前选择的相同ROI获得。因此，进行如步骤5.9（ 图 3C ）所述的整个细胞荧光分布的人口统计学分析。该分析清楚地显示了YFP-CheW定位的类似模式，其中YFP-CheW被单极定位在短细胞中并且双极定位在较长的细胞中。因此，指示细胞周期依赖性定位模式​​，其中YFP-CheW在最近分裂的细胞中单一定位（短），然后获得r在细胞周期（长细胞）之后再生于相反的极点，导致双极定位。对swarmer细胞进行了类似的分析（ 图4 ）。基于人群的分析显示，与游泳细胞类型相比，在Swellmer细胞中YFP-CheW的定位的主要差异。 YFP-CheW总是双极定位的（ 图4A ，黄色箭头），重要的是YFP-CheW也形成沿着细胞长度随机定位的簇（ 图4A ，橙色箭头）。因此，副溶血弧菌分化期间趋化性信号转导阵列的细胞内定位有重大变化。该实施例表明所描述的方法允许生产易于获得单细胞荧光显微镜的swarmer细胞群体。此外，它显示了用于图像分析的流水线允许细胞内组织的人口统计学分析副溶血弧菌swarmer细胞。 <img alt="图1" src="/files/ftp_upload/55842/55842fig1.jpg" /> 图1:Swarmer分化诱导和细胞显微镜成像的制备。简化可视化的工作流程以产生副溶血弧菌 swarmer细胞和制备显微镜幻灯片用于单个swarmer细胞的显微镜分析。 （ A ）将1μLOD HI = 0.8细胞培养的副溶血弧菌在HI琼脂积聚板的中央。 （ B ）孵育点直至干燥，细胞附着在琼脂表面。 （ C ）用透明胶带密封培养皿。将板在24℃孵育16小时。 （ D ）从群集的外缘小心切除一小块HI琼脂。 （ E ）将切口转移到微镜上振荡琼脂糖垫与swaromer细胞面向琼脂糖垫。短暂孵育30秒后，将细胞浸泡在琼脂糖垫上。 （ F ）小心地从琼脂糖垫中除去切除的琼脂，并将显微镜盖玻片放在上面。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55842/55842fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2" src="/files/ftp_upload/55842/55842fig2.jpg" /> 图2:副溶血性弧菌群落中细胞的组织。 （ A ）副溶血弧菌 swarmer殖民地的立体显微镜。上面的面板以增加的放大倍率显示群集群的外边缘。底部面板以增加的放大倍数显示中心和边缘之间的群集的区域。群星从周边延伸出来群集。 （ B ）代表性的DIC显微镜图像的swarmer殖民地边缘（上图）和中心与外围（底板）之间的区域从细胞从群体的各个指定位置转移后。如步骤3.8-3.11所述，将群发直接印刷在显微镜琼脂糖垫上。首先将从中心和周边区域的细胞从群集中刮下来并重新悬浮在LB肉汤中，然后再点到显微镜琼脂糖垫上。刻度棒=7.5μm。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55842/55842fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图3" src="/files/ftp_upload/55842/55842fig3.jpg" /> 图3:副溶血弧菌游泳细胞中趋化性数组的定位。 （ A ）DIC和异位表达YFP-CheW的野生型副溶血弧游泳细胞的荧光显微镜图像。 （ B ）通过绘制来自细胞极点的焦点距离作为游泳细胞中的细胞长度的函数来显示YFP-CheW焦点分布的图。 （ C ）表达YFP-CheW的副溶血性游泳细胞的荧光强度分布的人口统计学分析。细胞按细胞长度排序并显示相对荧光强度。刻度棒=5μm。图B和C适用于参考文献9 。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55842/55842fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图4" src="/files/ftp_upload/55842/55842fig4.jpg" /> 图4:副溶血性弧菌细胞中趋化性数组的定位。 </str（ A ）异位表达YFP-CheW的野生型副溶血弧菌swarmer细胞的DIC和荧光显微镜图像。 （ B ）通过在细胞中绘制来自细胞极的焦点距离作为细胞长度的函数来显示YFP-CheW焦点分布的图。 （ C ）表达YFP-CheW的副溶血性Tharivactus swarmer细胞的荧光强度谱的人口统计学分析。细胞按细胞长度排序并显示相对荧光强度。刻度棒=5μm。面板B和C适用于参考9 。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55842/55842fig4large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

Watch this Scientific Journal Video about Induction of Cellular Differentiation and Single Cell Imaging of Vibrio parahaemolyticus Swimmer and Swarmer Cells at JoVE.com

Induction of Cellular Differentiation and Single Cell Imaging of Vibrio parahaemolyticus Swimmer and Swarmer Cells

该协议允许单细胞显微镜检查差异不同的副溶血弧菌游泳运动员和swarmer细胞。该方法产生容易获得单细胞分析的swarmer细胞群，并涵盖细胞培养物的制备，诱导分子分化，样品制备和图像分析。

诱导细胞分化和单细胞成像<em&gt;副溶血性弧菌</em游泳者和Swarmer细胞

The ability to study the intracellular localization of proteins is essential for the understanding of many cellular processes. In turn, this requires the ...

induction-cellular-differentiation-single-cell-imaging-vibrio

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

Induction of Cellular Differentiation and Single Cell Imaging of Vibrio parahaemolyticus Swimmer and Swarmer Cells

9.3K 次观看.  Max Planck Institute for Terrestrial Microbiology. 该协议允许单细胞显微镜检查差异不同的副溶血弧菌游泳运动员和swarmer细胞。该方法产生容易获得单细胞分析的swarmer细胞群，并涵盖细胞培养物的制备，诱导分子分化，样品制备和图像分析。 

视频: Induction of Cellular Differentiation and Single Cell Imaging of Vibrio parahaemolyticus Swimmer and Swarmer Cells

Visualisation and Quantification of Intracellular Interactions of Neisseria meningitidis and Human &alpha;-actinin by Confocal Imaging

The Opc protein of Neisseria meningitidis (Nm, meningococcus) is a surface-expressed integral outer membrane protein, which can act as an adhesin and an effective invasin for human epithelial and endothelial cells. We have identified endothelial surface-located integrins as major receptors for Opc, a process which requires Opc to first bind to integrin ligands such as vitronectin and via these to the cell-expressed receptors1. This process leads to bacterial invasion of endothelial cells2. More recently, we observed an interaction of Opc with a 100kDa protein found in whole cell lysates of human cells3. We initially observed this interaction when host cell proteins separated by electrophoresis and blotted on to nitrocellulose were overlaid with Opc-expressing Nm. The interaction was direct and did not involve intermediate molecules. By mass spectrometry, we established the identity of the protein as &alpha;-actinin. As no surface expressed &alpha;-actinin was found on any of the eight cell lines examined, and as Opc interactions with endothelial cells in the presence of serum lead to bacterial entry into the target cells, we examined the possibility of the two proteins interacting intracellularly. For this, cultured human brain microvascular endothelial cells (HBMECs) were infected with Opc-expressing Nm for extended periods and the locations of internalised bacteria and &alpha;-actinin were examined by confocal microscopy. We observed time-dependent increase in colocalisation of Nm with the cytoskeletal protein, which was considerable after an eight hour period of bacterial internalisation. In addition, the use of quantitative imaging software enabled us to obtain a relative measure of the colocalisation of Nm with &alpha;-actinin and other cytoskeletal proteins. Here we present a protocol for visualisation and quantification of the colocalisation of the bacterium with intracellular proteins after bacterial entry into human endothelial cells, although the procedure is also applicable to human epithelial cells.

Visualisation and Quantification of Intracellular Interactions of Neisseria meningitidis and Human &#945;-actinin by Confocal Imaging

Neisseria meningitidis (Nm), a gram negative human-specific respiratory pathogen, can bind to human &alpha;-actinin. Here we present a protocol for visualisation of colocalisation of the bacterium with intracellular &alpha;-actinin after bacterial entry into human brain microvascular endothelial cells (HBMECs).

胞内相互作用的可视化和量化脑膜炎奈瑟菌和人类α-辅肌动蛋白共聚焦成像

The Opc protein of Neisseria meningitidis (Nm, meningococcus) is a surface-expressed integral outer membrane protein, which can act as an adhesin and an ...

科研

免疫与感染

Colonization of Euprymna scolopes Squid by Vibrio fischeri

Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no fitness consequence (commensal), or be beneficial (mutualistic). While much attention has been given to pathogenic interactions, little is known about the processes that dictate the reproducible acquisition of beneficial/commensal bacteria from the environment. The light-organ mutualism between the marine Gram-negative bacterium V. fischeri and the Hawaiian bobtail squid, E. scolopes, represents a highly specific interaction in which one host (E. scolopes) establishes a symbiotic relationship with only one bacterial species (V. fischeri) throughout the course of its lifetime6,7. Bioluminescence produced by V. fischeri during this interaction provides an anti-predatory benefit to E. scolopes during nocturnal activities8,9, while the nutrient-rich host tissue provides V. fischeri with a protected niche10. During each host generation, this relationship is recapitulated, thus representing a predictable process that can be assessed in detail at various stages of symbiotic development. In the laboratory, the juvenile squid hatch aposymbiotically (uncolonized), and, if collected within the first 30-60 minutes and transferred to symbiont-free water, cannot be colonized except by the experimental inoculum6. This interaction thus provides a useful model system in which to assess the individual steps that lead to specific acquisition of a symbiotic microbe from the environment11,12.
Here we describe a method to assess the degree of colonization that occurs when newly hatched aposymbiotic E. scolopes are exposed to (artificial) seawater containing V. fischeri. This simple assay describes inoculation, natural infection, and recovery of the bacterial symbiont from the nascent light organ of E. scolopes. Care is taken to provide a consistent environment for the animals during symbiotic development, especially with regard to water quality and light cues. Methods to characterize the symbiotic population described include (1) measurement of bacterially-derived bioluminescence, and (2) direct colony counting of recovered symbionts.

Colonization of Euprymna scolopes Squid by Vibrio fischeri

The method outlines the procedure by which the Hawaiian bobtail squid, Euprymna scolopes and its bacterial symbiont, Vibrio fischeri, are raised separately and then introduced to allow for specific colonization of the squid light organ by the bacteria. Colonization detection by bacterially-derived luminescence and by direct colony counting are described.

定植 Euprymna scolopes鱿鱼由弧菌鲵</em

Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no ...

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5.
Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8.
Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.

We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

定量高通量单细胞的细胞毒性T细胞的测定

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-&gamma; receptor 1) for the loading of monoclonal antibodies (mAb)12. In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR+ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of &gamma;-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-2113. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

T cells expressing a CD19-specific chimeric antigen receptor (CAR) are infused as investigational treatment of B-cell malignancies in our first-in-human gene therapy trials. We describe genetic modification of T cells using the Sleeping Beauty (SB) system to introduce CD19-specific CAR and selective propagation on designer CD19+ artificial antigen presenting cells.

临床应用<em&gt;睡美人</em和人工抗原呈递细胞基因修饰T细胞从外设和脐带血中

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer  a biologic product with the potential for ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

隔离和幼稚的CD4 T细胞的Th17细胞分化

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell interaction known as the immunological synapse. F-actin is also speculated to play a role in regulating molecular distributions at the membrane of cells including sub-membranous vesicle dynamics and protein clustering. While standard light microscope techniques allow generalized and diffraction-limited observations to be made, many cellular and molecular events including clustering and molecular flow occur in populations at length-scales far below the resolving power of standard light microscopy. By combining total internal reflection fluorescence with the super resolution imaging method structured illumination microscopy, the two-dimensional molecular flow of F-actin at the immune synapse of T cells was recorded. Spatio-temporal image correlation spectroscopy (STICS) was then applied, which generates quantifiable results in the form of velocity histograms and vector maps representing flow directionality and magnitude. This protocol describes the combination of super-resolution imaging and STICS techniques to generate flow vectors at sub-diffraction levels of detail. This technique was used to confirm an actin flow that is symmetrically retrograde and centripetal throughout the periphery of T cells upon synapse formation.

To investigate flow velocities and directionality of filamentous-actin at the T cell immunological synapse, live-cell super-resolution imaging is combined with total internal reflection fluorescence and quantified with spatio-temporal image correlation spectroscopy.

皮层肌动蛋白流通过结构照明显微镜数据的时空图像相关光谱量化性T细胞

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell ...

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the majority of Vibrio-associated infections. Thus, accurate differentiation among Vibrio spp. and detection of V. parahaemolyticus is critically important to ensure the safety of our food supply. Although molecular techniques are increasingly common, culture-depending methods are still routinely done and they are considered standard methods in certain circumstances. Hence, a novel chromogenic agar medium was tested with the goal of providing a better method for isolation and differentiation of clinically relevant Vibrio spp. The protocol compared the sensitivity, specificity and detection limit for the detection of V. parahaemolyticus between the new chromogenic medium and a conventional medium. Various V. parahaemolyticus strains (n=22) representing diverse serotypes and source of origins were used. They were previously identified by Food and Drug Administration (FDA) and Centers for Disease Control and Prevention (CDC), and further verified in our laboratory by tlh-PCR. In at least four separate trials, these strains were inoculated on the chromogenic agar and thiosulfate-citrate-bile salts-sucrose (TCBS) agar, which is the recommended medium for culturing this species, followed by incubation at 35-37 &#176;C for 24-96 hr. Three V. parahaemolyticus strains (13.6%) did not grow optimally on TCBS, nonetheless exhibited green colonies if there was growth. Two strains (9.1%) did not yield the expected cyan colonies on the chromogenic agar. Non-V. parahaemolyticus strains (n=32) were also tested to determine the specificity of the chromogenic agar. Among these strains, 31 did not grow or exhibited other colony morphologies. The mean recovery of V. parahaemolyticus on the chromogenic agar was ~96.4% relative to tryptic soy agar supplemented with 2% NaCl. In conclusion, the new chromogenic agar is an effective medium to detect V. parahaemolyticus and to differentiate it from other vibrios.

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

Detection and isolation of clinically relevant Vibrio species require selective and differential culture media. This study evaluated the ability of a new chromogenic medium to detect and identify V. parahaemolyticus and other related species. The new medium was found to have better sensitivity and specificity than the conventional medium.

对于检测到的敏感性和特异性显色琼脂培养基发展<em&gt;副溶血性弧菌</em&gt;及其他<em&gt;弧菌</em&gt;物种

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the ...

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 ...

Assessing Cellular Stress and Inflammation in Discrete Oxytocin-secreting Brain Nuclei in the Neonatal Rat Before and After First Colostrum Feeding

The goal of this protocol is to isolate oxytocin-receptor rich brain nuclei in the neonatal brain before and after first colostrum feeding. The expression of proteins known to respond to metabolic stress was measured in brain-nuclei isolates using Western blotting. This was done to assess whether metabolic stress-induced nutrient insufficiency in the body triggered neuronal stress. We have previously demonstrated that nutrient insufficiency in neonates elicits metabolic stress in the gut. Furthermore, colostrum oxytocin modulates cellular stress response, inflammation, and autophagy markers in newborn rat gut villi prior to and after first feed. Signaling protein markers associated with the endoplasmic reticulum stress [ER chaperone binding immunoglobulin protein (BiP), eukaryotic translation initiation factor 2A (eIF2a), and eIF2a kinase protein kinase R (p-PKR)], as well as two inflammation-signaling proteins [nuclear factor-&#954;B (NF-kB) and inhibitor &#954;B (IkB)], were measured in newborn brain nuclei [nucleus of the solitary tract (NTS), paraventricular nucleus (PVN), supra-optic nucleus (SON), cortex (CX), striatum nuclei (STR), and medial preoptic nucleus (MPO)] before the first feed (unprimed by colostrum) and after the start of nursing (primed by colostrum). Expression of BiP/GRP78 and p-eIF2a were upregulated in unprimed and downregulated in primed NTS tissue. NF-kB was retained (high) in the CX, STR, and MPO cytoplasm, whereas NF-kB was lower and unchanged in NTS, PVN, and SON in both conditions. The collective BiP and p-eIF2 findings are consistent with a stress response. eIf2a was phosphorylated by dsRNA dependent kinase (p-PKR) in the SON, CX, STR, and MPO. However, in the NTS (and to a lesser extent in PVN), eIf2a was phosphorylated by another kinase, general control nonderepressible-2 kinase (GCN2). The stress-modulating mechanisms previously observed in newborn gut enterocytes appear to be mirrored in some OTR-rich brain regions. The NTS and PVN may utilize a different phosphorylation mechanism (under nutrient deficiency) from other regions and be refractory to the impact of nutrient insufficiency. Collectively, this data suggests that brain responses to nutrient insufficiency stress are offset by signaling from colostrum-primed enterocytes.

Here, we present a protocol to isolate brain nuclei in the neonatal rat brain in conjunction with first colostrum feeding. This technique allows the study of nutrient insufficiency stress in the brain as modulated by enterocyte signaling.

新生儿初乳喂养前后离散催产素分泌脑核细胞应激与炎症的评估

The goal of this protocol is to isolate oxytocin-receptor rich brain nuclei in the neonatal brain before and after first colostrum feeding. The expression ...

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 ...