这项工作是由青年研究者的研究计划，从维尔茨堡大学传染病研究中心（ZINF）。胡安·Ç加西亚贝坦库尔是从维尔茨堡大学生命科学院研究生院（GSLS）博士研究员。

1。标签B。枯草和生物膜形成实验<ol><li>通过PCR扩增感兴趣的基因的启动子区域。我们的P TAPA，负责生产的的TASA基质蛋白26基因启动子的克隆为例。克隆带够TAPA到pkm008向量（Rudner实验室，哈佛医学院，波士顿，美国）（图2）。 </li><li>线性质粒，通过消化酶（酶建议，XhoI位）。 </li><li> 在 B诱导自然能力枯草芽孢杆菌菌株按照协议，先前由Harwood一步切割27 168。 </li><li>添加到文化主管细胞的线性质粒和两个小时的潜伏期后，奇霉素抗性选择。 </li><li>株获得插入到B 的的中性amyE轨迹的构造双重组枯草芽孢杆菌 （图4）。要创建双标记菌株，融入中性轨迹LACA使用的质粒pDR183我们在图3中给出的第二个记者。插入记者，以上我们描述了克隆的记者在pKM008的插入使用相同的技术。 </li><li>记者从168菌株转移到NCIB3610是能够形成生物膜。使用SPP1噬菌体转导的协议28,29。在TY培养基增长捐助株（LB +10 mM的硫酸镁4 +10微米硫酸锰4）。混合培养液100μL噬菌体股票稀释200。孵育30分钟后加入3毫升软琼脂，并允许噬菌体光晕出现在37°C。 </li><li>收集软琼脂。离心机，并通过上清认为注射器0.22微米的过滤器。使用此上清感染在TY培养基上生长的受体菌株的文化。加入30微升至10毫升稀释的文化1:10。孵育30分钟和24小时的潜伏期后选择抗生素耐药性。 </li><li>硒LECT殖民地和37一夜之间生长在LB°C。 </li><li>坚实的生物膜诱导MSgg的1.5％，琼脂30介质上，发现3μL过夜培养。让细胞在72小时生长在30°C（图3）。经过三天的增长，琼脂表面的表面的MSgg琼脂上形成的生物膜开发一个复杂的形态结构。 </li></ol> 2。生物膜分散和细胞固定<ol><li>表面，MSgg琼脂用牙签或镊子取出生物膜形式。生物膜的一致性，应该让你在一块琼脂表面剥离它。 </li><li>将在3毫升PBS缓冲生物膜和分散通过吸管或针头重复通过生物膜。此外，生物膜可以用温和的超声分散。轻度超声需要12个脉冲输出3和0.7秒的幅度。 </li><li>修正样品前单细胞分析。 resuspenD 300μL 整整七分钟，在4％多聚甲醛溶液，室温孵育1毫升细胞悬液。 4％多聚甲醛溶液的组成： 2克多聚甲醛 PBS缓冲液50毫升 4μL为10 N氢氧化钠 过滤的解决方案，通过0.22微米的过滤器和等分</li><li>洗净后，PBS缓冲液中的固定三次的细胞和悬浮在300μLPBS缓冲。 </li></ol> 3。荧光显微镜<ol><li>倒入200μL琼脂糖显微镜幻灯片0.8％，并仔细盖上另一张幻灯片。 2分钟后取出上层幻灯片，轻轻地获得琼脂糖层底部的幻灯片。 </li><li>现货2μL琼脂糖层表面上的固定细胞，用显微镜盖玻片覆盖。 </li><li>放置在荧光显微镜的样品。我们用荧光显微镜徕卡DMI6000B与徕卡CRT6000 iIluminat的装备离子系统。 YFP的过滤器例如：BP500/20，EM：BP535/30和CFP资格的防爆：BP426/20，EM：480/40 </li><li>公开您的样本 50-200毫秒之间激发荧光。设置激发期内根据阴性对照，这表明在实验选定的条件没有荧光。 </li><li>有关荧光形象与明亮的领域获得相同的图像。合并在一张照片中的两个图像。从流荧光显微镜用单标记的应变窝藏记者带够TAPA-YFP的代表在图6得到的结果。 </li></ol> 4。单细胞用流式细胞仪定量<ol><li>固定细胞，用温和的超声分散的样品。超声波清洗的样品进行5 2系列12脉冲输出和0.7秒的幅度，分散成单个细胞团块，而导致细胞裂解。通过光镜确认细胞扩散的效率。 </李&gt; <li>流式细胞仪分析前的样品在PBS缓冲液稀释1:100。我们用流式细胞仪的BD流式细胞仪广东第二。对于YFP的荧光，再加上530/30日过滤器使用488纳米的激光激发。对CFP荧光，再加上一个408/40过滤器使用405纳米的激光激发。 </li><li>流式细胞仪机校准与两个阴性对照。 PBS的细胞悬浮液中没有缓冲区的样本应作为负粒子的大小，流式细胞仪检测到控制。与记者无荧光标记的样本应作为阴性对照的荧光，流式细胞仪灵敏度。 </li><li>将与记者在流式细胞仪的荧光标记的样本。对于每个样本，分析至少50000之间的300和3000事件每秒的流量事件。 </li><li>使用流式细胞仪，DIVA软件（BD Biosciences公司）采集数据和分析它使用FlowJo 8.5.2软件。是指荧光信号，表明控制没有荧光。 </li><li>目前监测的数据在两轴图形应变单标记的荧光信号。绘制在X轴和表达的荧光在Y轴的不同层次的细胞数量检测荧光信号。用单标记的应变窝藏记者带够TAPA-YFP的代表在图8流式细胞仪分析得到的结果。 </li><li>提出的双标在三个轴的平面应变监测荧光信号的数据。画出每一个在X和Y轴（例如，GFP将在X轴和Y轴CFP资格测量）监测通道的荧光信号。情节细胞表达每个记者和呈现轮廓等值线，这将是垂直于平面的纸张（图9和10），在Z轴。 </li></ol> 4。代表结果当B。枯草增长一盘的B观察3天的潜伏期后，在30°C 30 iofilm诱导培养基MSgg的生物膜形成。生物膜显示了很强的一致性，它可以在一块琼脂表面去皮。此外，生物膜，显示了一个复杂的形态结构是指示鲜明参与细胞亚群（图5）。例如，在细胞外基质在殖民地表面上的皱纹形成的生物膜结果。此功能可与矩阵生产者19亚群的分化。同样，提高生物膜表面上的天线结构是表明sporulating细胞亚群的存在，因为孢子在这些结构30心尖区本地化。 细胞分化的可视化的例子，在一个单标记和双标记的菌株，用荧光显微镜ŕepresented在图6和7，分别为。单标记的应变藏着荧光记者带够TAPA-CFP资格是在基质细胞亚群表示。这种亚群是负责产生和分泌细胞外基质构成生物膜（图6）。双标记菌株窝藏记者带够26 TAPA-CFP资格和额外的记者带够srfAA-YFP的31。这第二个记者，允许监视亚细胞负责分泌的信号分子的表面活性，从而触发信号级联矩阵生产者的分化（图7）。亚群矩阵生产者都是假的蓝色，而亚表面活性生产者是假的黄色。 流式细胞仪分析用单标记的应变窝藏记者带够TAPA-YFP的图8。未经处理的对照菌株不包庇任何荧光蛋白基因，表明一个单一的人口相对低荧光。没带够TAPA-YFP的非生物膜的诱导条件细胞窝藏区分亚矩阵生产者和整个人口呈相对低的荧光。在生物膜的诱导条件下，具有相对高的荧光的细胞亚群发生，作为一个肩膀观察到23低相对荧光峰的权利。 从流式细胞仪采用双标记菌株的分析得到的结果表示在图9和10。图9监视矩阵生产者和表面活性生产者使用双标应变带够，磷srfAA-YFP的TAPA-CFP资格的亚群。作为背景荧光的控制，我们使用没有窝藏任何荧光蛋白基因的菌株。下一步，我们发现在每个荧光通道，每个亚矩阵生产者和表面活性生产者，使用单标记菌株<html> S为控制。双标应变带够，磷srfAA-YFP的TAPA-CFP资格表明了两个高层次的荧光记者表达的细胞亚群。每个人口诬陷，显示有15个专门的细胞两个亚群之间的记者表达没有重叠。同样，使用双标流式细胞仪分析应变带够，磷TAPA-YFP的SKF-YFP的是在图10。基因SKF记者监视食人族32，已被描述的协调与亚矩阵生产者24区分亚群的分化。在这种情况下，双标应变单一的表达YFP的和CFP的荧光细胞亚群。这表明，无论是协调在同一亚群激活细胞分化途径。 1.JPG“/&gt; 图1。实验的总体方案。该协议分为三个主要步骤。第一步需要标注B 的应变记者枯草融合，监控亚利益。第二，成长标记菌株在生物膜的诱导条件。第三，分散生物膜，并进行单细胞的人口使用荧光显微镜和流式细胞仪分析。 <img alt="图2" src="/files/ftp_upload/3796/3796fig2.jpg" /> 图2。这个向量的示意图整合载体pKM008的。集成到双重组中性轨迹amyE记者感兴趣的融合。利息（TAPA）的启动子克隆到使用EcoRI和酶切的酶切位点的载体。然后，CFP基因的表达，是控制下的子带够TAPA。在orientat离子基因质粒作为一个箭头表示。 <img alt="图3" src="/files/ftp_upload/3796/3796fig3.jpg" /> 图3。这个载体整合载体pDR183示意图。双重组整合到中立位点LACA记者感兴趣的融合。 EcoRI和 BamHI酶切位点克隆到载体利息（TAPA - CFP）的融合。作为一个箭头表示基因质粒的方向。 <img alt="图4" src="/files/ftp_upload/3796/3796fig4.jpg" /> 图4。记者一体化的计划，到B 的染色体由双重组枯草 。 （一）B （二） 枯草染色体有两个中性位点，amyE和LACA，结合记者的融合，而不影响生物膜的发展。 </s豪华型&gt;双重组pKM008的过程。线性质粒整合到B 的基因由双重组枯草 。记者融合集成到中性轨迹在一个稳定的方式。 <img alt="图5" src="/files/ftp_upload/3796/3796fig5.jpg" /> 图5。 B 的生物膜形成枯草 NCIB3610应变B. bioflm形成过程枯草 NCIB 3610时，越来越多在三天的生物膜诱导培养基MSgg的30°C。每12h生物膜发展的连续照片拍摄。 <img alt="图6" src="/files/ftp_upload/3796/3796fig6.jpg" /> 图6。在荧光显微镜下的矩阵生产商亚群的可视化从B 的生物膜样品。 枯草带够TAPA - CFP是固定的，在荧光显微镜下检查。 200毫秒的荧光激发证明散发高于其他细胞的荧光细胞亚群。我们认为作为基质产生细胞亚群亚群。比例尺为3μm。 <img alt="图7" src="/files/ftp_upload/3796/3796fig7.jpg" /> 图7。从生物膜样品的B. 亚矩阵生产者和荧光显微镜下的表面活性生产者的可视化。 枯草带够TAPA-CFP供磷srfAA-YFP双标应变是固定的，在荧光显微镜下检查。 250毫秒证明两个排放高于其他细胞的荧光细胞亚群的曝光时间。一个亚群表达YFP的独家使用YFP的通道（假黄色）检测。这是亚表面活性生产者。另一个亚群表达CFP和它是专门检测在CFP陈荫罴<html> EL。这是亚矩阵生产者。比例尺为3μm。 <img alt="图8" src="/files/ftp_upload/3796/3796fig8.jpg" /> 图8。量化矩阵生产者使用的2-D流式细胞仪。亚群分散从B 的生物膜的细胞枯草带够TAPA-YFP进行了监测，用流式细胞仪。流式细胞仪计数50.000事件，每个事件的荧光信号监测。计数细胞数绘制在绘制在X轴Y轴，而YFP的信号强度。在LB培养基中获得细胞生长非生物膜诱发条件。在MSgg媒介获得生物膜诱导条件细胞生长。亚矩阵生产者的区别只在生物膜的诱导条件。这个数字是改编自洛佩斯等。，国家科学院院刊（2009）106 :280-285。 s/ftp_upload/3796/3796fig9.jpg“/&gt; 图9。亚矩阵生产者和表面活性生产者使用的3-D流式细胞仪定量监测通道的荧光信号。在X轴（YFP的）和Y轴（CFP资格）。 Z轴测量细胞表达每个记者的数量，并进行量化轮廓等值线垂直的平面纸张。在这个实验中监视的事件数量为50.000事件。左上小组提出的背景荧光窝藏无荧光蛋白基因的控制。右上面板检测亚表面活性生产者YFP的荧光通道（黄色框），使用一个单一的标记菌株带够srfAA YFP的。左下角面板检测亚矩阵生产者使用单一标记的应变带够TAPA-CFP资格CFP荧光通道（蓝 ​​色框）。双标记的应变带够TAPA &lt;/ EM&gt; CFP资格，磷srfAA-YFP的监测在右下角面板。它表明在黄色和蓝色镜框的两个亚群。这个数字是改编自洛佩斯等 ，基因和发展（2009）23 :1631-1638。 <img alt="图10" src="/files/ftp_upload/3796/3796fig10.jpg" /> 图10。量化矩阵使用的3-D流式细胞仪的生产商和食人族亚群的监测通道的荧光信号。在X轴（YFP的）和Y轴（CFP资格）。在这个实验中监视的事件数量为50.000。右上面板检测亚YFP的荧光通道（黄色框）在一个单一标记的应变带够SKF-YFP的食人族。左下角面板检测基质生产者的亚单标记的应变带够TAPA-CFP资格CFP荧光通道（蓝 ​​色框）。在Double标记应变带够，磷SKF-YFP的TAPA-CFP资格显示对角线的X和Y轴（绿色框）只有一个细胞亚群。这亚群检测YFP的渠道和CFP，因为它同时表示，两名记者。洛佩斯等，基因和发展（2009）23 :1631-1638。

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B., Losick, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+population+heterogeneity+during+growth+of+Bacillus+subtilis.">Cell population heterogeneity during growth of Bacillus subtilis.</a> Genes Dev. 19, 3083-3094 (2005).</li><li>Lopez, D., Vlamakis, H., Losick, R., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paracrine+signaling+in+a+bacterium.">Paracrine signaling in a bacterium.</a> Genes Dev. 23, 1631-1638 (2009).</li><li>Veening, J. W., Smits, W. K., Hamoen, L. W., Jongbloed, J. D., Kuipers, O. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+differential+gene+expression+by+improved+cyan+fluorescent+protein+and+yellow+fluorescent+protein+production+in+Bacillus+subtilis.">Visualization of differential gene expression by improved cyan fluorescent protein and yellow fluorescent protein production in Bacillus subtilis.</a> Appl. Environ. Microbiol. 70, 6809-6815 (2004).</li><li>Veening, J. W., Smits, W. K., Hamoen, L. W., Kuipers, O. P. <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+analysis+of+gene+expression+patterns+of+competence+development+and+initiation+of+sporulation+in+Bacillus+subtilis+grown+on+chemically+defined+media.">Single cell analysis of gene expression patterns of competence development and initiation of sporulation in Bacillus subtilis grown on chemically defined media.</a> J. Appl. Microbiol. 101, 531-541 (2006).</li><li>Veening, J. W., Kuipers, O. P., Brul, S., Hellingwerf, K. J., Kort, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+phosphorelay+perturbations+on+architecture,+sporulation,+and+spore+resistance+in+biofilms+of+Bacillus+subtilis.">Effects of phosphorelay perturbations on architecture, sporulation, and spore resistance in biofilms of Bacillus subtilis.</a> J. Bacteriol. 188, 3099-3109 (2006).</li><li>Vlamakis, H., Aguilar, C., Losick, R., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+cell+fate+by+the+formation+of+an+architecturally+complex+bacterial+community.">Control of cell fate by the formation of an architecturally complex bacterial community.</a> Genes Dev. 22, 945-953 (2008).</li><li>Stewart, P. S., Franklin, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+heterogeneity+in+biofilms.">Physiological heterogeneity in biofilms.</a> Nat. Rev. Microbiol. 6, 199-210 (2008).</li><li>Veening, J. W., Smits, W. K., Kuipers, O. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bistability,+epigenetics,+and+bet-hedging+in+bacteria.">Bistability, epigenetics, and bet-hedging in bacteria.</a> Annu. Rev. Microbiol. 62, 193-210 (2008).</li><li>Aguilar, C., Vlamakis, H., Guzman, A., Losick, R., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KinD+is+a+checkpoint+protein+linking+spore+formation+to+extracellular-matrix+production+in+Bacillus+subtilis+biofilms.">KinD is a checkpoint protein linking spore formation to extracellular-matrix production in Bacillus subtilis biofilms.</a> MBio. 1, (2010).</li><li>Lopez, D., Fischbach, M. A., Chu, F., Losick, R., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structurally+diverse+natural+products+that+cause+potassium+leakage+trigger+multicellularity+in+Bacillus+subtilis.">Structurally diverse natural products that cause potassium leakage trigger multicellularity in Bacillus subtilis.</a> Proc. Natl. Acad. Sci. U.S.A. 106, 280-285 (2009).</li><li>Lopez, D., Vlamakis, H., Losick, R., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cannibalism+enhances+biofilm+development+in+Bacillus+subtilis.">Cannibalism enhances biofilm development in Bacillus subtilis.</a> Mol. Microbiol. 74, 609-618 (2009).</li><li>Arima, K., Kakinuma, A., Tamura, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surfactin,+a+crystalline+peptidelipid+surfactant+produced+by+Bacillus+subtilis:+isolation,+characterization+and+its+inhibition+of+fibrin+clot+formation.">Surfactin, a crystalline peptidelipid surfactant produced by Bacillus subtilis: isolation, characterization and its inhibition of fibrin clot formation.</a> Biochem. Biophys. Res. Commun. 31, 488-494 (1968).</li><li>Romero, D., Vlamakis, H., Losick, R., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+accessory+protein+required+for+anchoring+and+assembly+of+amyloid+fibres+in+B.+subtilis+biofilms.">An accessory protein required for anchoring and assembly of amyloid fibres in B. subtilis biofilms.</a> Mol. Microbiol. 80, 1155-1168 (2011).</li><li>Hardwood, C. R., Cutting, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Biological Methods for Bacillus. , (1990).</li><li>Novick, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+systems+in+staphylococci.">Genetic systems in staphylococci.</a> Methods Enzymol. 204, 587-636 (1991).</li><li>Yasbin, R. E., Young, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transduction+in+Bacillus+subtilis+by+bacteriophage+SPP1.">Transduction in Bacillus subtilis by bacteriophage SPP1.</a> J. Virol. 14, 1343-1348 (1974).</li><li>Branda, S. S., Gonzalez-Pastor, J. E., Ben-Yehuda, S., Losick, R., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fruiting+body+formation+by+Bacillus+subtilis.">Fruiting body formation by Bacillus subtilis.</a> Proc. Natl. Acad. Sci. U.S.A. 98, 11621-11626 (2001).</li><li>Nakano, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=srfA+is+an+operon+required+for+surfactin+production,+competence+development,+and+efficient+sporulation+in+Bacillus+subtilis.">srfA is an operon required for surfactin production, competence development, and efficient sporulation in Bacillus subtilis.</a> J. Bacteriol. 173, 1770-1778 (1991).</li><li>Gonzalez-Pastor, J. E., Hobbs, E. C., Losick, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cannibalism+by+sporulating+bacteria.">Cannibalism by sporulating bacteria.</a> Science. 301, 510-513 (2003).</li><li>Aguilar, C., Vlamakis, H., Losick, R., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thinking+about+Bacillus+subtilis+as+a+multicellular+organism.">Thinking about Bacillus subtilis as a multicellular organism.</a> Curr. Opin. Microbiol. 10, 638-643 (2007).</li><li>Shapiro, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thinking+about+bacterial+populations+as+multicellular+organisms.">Thinking about bacterial populations as multicellular organisms.</a> Annu. Rev. Microbiol. 52, 81-104 (1998).</li></ol>

我们什么都没有透露。

事实上，细菌群落表明细胞亚群的表达一组特定的基因证据33,34微生物群落的复杂性。本议定书应帮助，以确定是否对任何感兴趣的基因的表达仅限于微生物群落，特别是一个专门的细胞亚群。这些亚群的可视化需要新技术的发展，因为传统的方法来监测基因表达微阵列基因表达分析率水平，整个微生物群落内的微生物群落的基因表达的波动一般失手。 荧光显微镜和?...

<table border="1"><tbody><tr><td> 技术 </td><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td rowspan="11"> MSgg组成</td><td>磷酸钾5MM </td><td>罗斯</td><td> 6878 </td></tr><tr><td>公开资讯观测站100MM </td><td> Sigma-Aldrich公司</td><td> M1254 </td></tr><tr><td>氯化镁2MM </td><td>罗斯</td><td> 2189.1 </td></tr><tr><td>钙氯化物70​​0μM </td><td>罗斯</td><td> A119.1 </td></tr><tr><td>三氯化铁50μM </td><td> Sigma-Aldrich公司</td><td> 157740 </td></tr><tr><td>锌氯1μM </td><td> applichem </td><td> A2076 </td></tr><tr><td>硫胺素2μM </td><td> Sigma-Aldrich公司</td><td> 74625 </td></tr><tr><td>甘油0.5％ </td><td>罗斯</td><td> 7533 </td></tr><tr><td>谷氨酸队友0.5％ </td><td> Sigma-Aldrich公司</td><td> 49621 </td></tr><tr><td>色氨酸为50μg/ml </td><td> Sigma-Aldrich公司</td><td> T0254 </td></tr><tr><td>苯丙氨酸为50μg/ml </td><td> Sigma-Aldrich公司</td><td> P2126 </td></tr><tr><td>细胞固定</td><td>多聚甲醛</td><td>罗斯</td><td> 0335 </td></tr><tr><td></td><td> 设备名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>超声</td><td>细胞Sonicator </td><td> bandelin </td><td> D - 1000 </td></tr><tr><td>荧光显微镜</td><td>荧光显微镜</td><td>徕卡</td><td> DMI6000B </td></tr><tr><td></td><td> 该软件的名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>荧光显微镜</td> &lt;TD&gt; ASAF </td><td>徕卡</td><td></td></tr><tr><td>流式细胞仪</td><td> FCASDiva </td><td>屋宇署</td><td></td></tr><tr><td>流式细胞仪</td><td> FlowJo </td><td> treestar </td><td></td></tr></tbody></table>

single-cell analysis of bacillus subtilis biofilms using fluorescence microscopy and flow cytometry

几乎所有的细菌1-6生物膜的形成是一个普遍的属性。当细菌形成生物膜，包裹在细胞外基质，大多在7-10的其他因素所构成的蛋白质和胞外多糖，。包裹内生物膜的微生物群落，往往显示11-17专门的细胞不同亚群的分化。这些亚群共存，并常常表现出时间和空间内的生物膜18-21组织。 在模式生物的枯草芽孢杆菌的生物膜的形成需要专门的细胞不同亚群的分化。其中，亚矩阵生产商，负责产生和分泌的生物膜的细胞外基质是必不可少的生物膜形成11,19。因此，矩阵生产者的分化是生物膜形成的标志在 B 枯草。 我们使用荧光记者可视化和量化亚矩阵生产者B 的生物膜枯草 15,19,22-24。具体来说，我们观察到亚矩阵生产者自产外信号表面活性25存在区别。有趣的是，表面活性是由亚矩阵生产者15不同的专门细胞亚群。 我们已详列于本报告必要的技术方法，可视化和量化矩阵生产者和表面活性生产者在枯草杆菌生物膜亚群。要做到这一点，基质生产和生产表面活性所需的基因插入染色体B 的荧光记者枯草 。记者们表示，只有在一个专门的细胞亚群。然后，亚能监测用荧光显微镜和流式细胞仪（见图1）。 专门的细胞不同亚群内的细菌多细胞群落共存的事实给了我们一个不同的角度对原核生物的基因表达调控。该协议解决了这一实验现象和它可以很容易地适应任何其他的工作模式，以澄清在微生物群落的基本表型异质性的分子机制。

Watch this Scientific Journal Video about Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry at JoVE.com

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

微生物生物膜一般由专门的细胞不同亚群构成。这些亚群的单细胞分析需要使用荧光记者。在这里，我们描述了一个协议，以可视化和监视几个subpopulationswithin<em&gt; B枯草</em&gt;生物膜，用荧光显微镜和流式细胞仪。

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 ...

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Research

JoVE Journal

Immunology and Infection

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

20.3K 次观看.  University of Würzburg. 微生物生物膜一般由专门的细胞不同亚群构成。这些亚群的单细胞分析需要使用荧光记者。在这里，我们描述了一个协议，以可视化和监视几个subpopulationswithin B枯草生物膜，用荧光显微镜和流式细胞仪。

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

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

流式细胞仪分析小鼠主动脉内流的免疫细胞

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

科研

免疫与感染

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see 1,2,3). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy).
Here, we provide a detailed description of a microscopy method used in several recent studies 4, 5, 6, 7, which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ.

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

This protocol provides a step-by-step procedure to monitor single cell behavior of different bacteria in time using automated fluorescence time-lapse microscopy. Furthermore, we provide guidelines how to analyze the microscopy images.

活细胞成像枯草芽孢杆菌肺炎链球菌使用自动定时显微镜

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not ...

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted as an important hallmark of cancer. Our research on breast cancer focuses on the active role that tumor infiltrating lymphocytes play in tumor progression and patient outcome. Toward this goal, we developed a methodology for the rapid isolation of intact lymphoid cells from normal and abnormal tissues in an effort to evaluate them proximate to their native state. Homogenates prepared using a mechanical dissociator show both increased viability and cell recovery while preserving surface receptor expression compared to enzyme-digested tissues. Furthermore, enzymatic digestion of the remaining insoluble material did not recover additional CD45+ cells indicating that quantitative and qualitative measurements in the primary homogenate likely genuinely reflect infiltrating subpopulations in the tissue fragment. The lymphoid cells in these homogenates can be easily characterized using immunological (phenotype, proliferation, etc.) or molecular (DNA, RNA and/or protein) approaches. CD45+ cells can also be used for subpopulation purification, in vitro expansion or cryopreservation. An additional benefit of this approach is that the primary tissue supernatant from the homogenates can be used to characterize and compare cytokines, chemokines, immunoglobulins and antigens present in normal and malignant tissues. This protocol functions extremely well for human breast tissues and should be applicable to a wide variety of normal and abnormal tissues.

This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45+ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.

一种简单快速议定书非酶离解新鲜人组织的浸润淋巴细胞的分析

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted ...

Prevention of Heat Stress Adverse Effects in Rats by Bacillus subtilis Strain

This study was designed to evaluate the protective effect of the Bacillus subtilis strain against complications related to heat stress. Thirty-two Sprague-Dawley rats were used in this study. Animals were orally treated twice a day for two days with B. subtilis BSB3 strain or PBS. The next day after the last treatment, each group was divided and two experimental groups (one treated with PBS and one treated with B. subtilis) were placed at 45 oC for 25 min. Two control groups stayed for 25 min at room temperature. All rats were euthanized and different parameters were analyzed in all groups. Adverse effects of heat stress are registered by the decrease of villi height and total mucosal thickness in the intestinal epithelium; translocation of bacteria from the lumen; increased vesiculation of erythrocytes and elevation of the lipopolysaccharides (LPS) level in the blood. The protective efficacy of treatment is evaluated by prevention of these side effects. The protocol was set up for the oral treatment of rats with bacteria for prevention of heat stress complications, but this protocol can be modified and used for other routes of administration and for analysis of different compounds.

Prevention of Heat Stress Adverse Effects in Rats by Bacillus subtilis Strain

We described a protocol for prevention of heat stress effects in rats by oral pre-treatment with beneficial bacteria. This protocol can be modified and used for various routes of administration and for analysis of different compounds.

热火预防应激在大鼠不利影响<em&gt;枯草芽孢杆菌</em&gt;应变

This study was designed to evaluate the protective effect of the Bacillus subtilis strain against complications related to heat stress. Thirty-two ...

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and resilience of Bacillus subtilis biofilms. First, methods are presented that select for small molecule inhibitors with biofilm-specific targets in order to separate the effect of the small molecule inhibitors on planktonic growth from their effect on biofilm formation. Next, we focus on how inoculation conditions affect the sensitivity of multicellular, floating B. subtilis cultures to small molecule inhibitors. The results suggest that discrepancies in the reported effects of such inhibitors such as D-amino acids are due to inconsistent pre-culture conditions. Furthermore, a recently developed protocol is described for evaluating the contribution of small molecule treatments towards biofilm resistance to antibacterial substances. Lastly, scanning electron microscopy (SEM) techniques are presented to analyze the three-dimensional spatial arrangement of cells and their surrounding extracellular matrix in a B. subtilis biofilm. SEM facilitates insight into the three-dimensional biofilm architecture and the matrix texture. A combination of the methods described here can greatly assist the study of biofilm development in the presence and absence of biofilm inhibitors, and shed light on the mechanism of action of these inhibitors.

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This study presents the development of reproducible methodologies to study biofilm inhibitors and their effects on Bacillus subtilis multicellularity.

方法学研究<em&gt;乙。枯草</em&gt;生物膜作为模型的表征小分子抑制剂生物膜

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

荧光激活细胞分选从小鼠骨髓浆树突状细胞的分离纯化

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

小鼠与人肺泡巨噬细胞的分离和体外培养

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can have pathological significance. An ideal method for examining alterations to monocytes is whole blood flow cytometry as the minimal handling of samples by this method limits artifactual cell activation. However, many different approaches are taken to gate the monocyte subsets leading to inconsistent identification of the subsets between studies. Here we demonstrate a method using whole blood flow cytometry to identify and characterize human monocyte subsets (classical, intermediate, and non-classical). We outline how to prepare the blood samples for flow cytometry, gate the subsets (ensure contaminating cells have been removed), and determine monocyte subset expression of surface markers &#8212; in this example M1 and M2 markers. This protocol can be extended to other studies that require a standard gating method for assessing monocyte subset proportions and monocyte subset expression of other functional markers.

Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory).

全血流式细胞术分析人单核细胞亚群的表征

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling and cellular processes that control efferocytosis. This quantification can be difficult to perform as apoptotic cells are often efferocytosed piecemeal, thus necessitating methods which can accurately delineate between the efferocytosed portion of an apoptotic target versus residual unengulfed cellular fragments. The approach outlined herein utilizes dual-labeling approaches to accurately quantify the dynamics of efferocytosis and efferocytic capacity of efferocytes such as macrophages. The cytosol of the apoptotic cell is labeled with a cell-tracking dye to enable monitoring of all apoptotic cell-derived materials, while surface biotinylation of the apoptotic cell allows for differentiation between internalized and non-internalized apoptotic cell fractions. The efferocytic capacity of efferocytes is determined by taking fluorescent images of live or fixed cells and quantifying the amount of bound versus internalized targets, as differentiated by streptavidin staining. This approach offers several advantages over methods such as flow cytometry, namely the accurate delineation of non-efferocytosed versus efferocytosed apoptotic cell fractions, the ability to measure efferocytic dynamics by live-cell microscopy, and the capacity to perform studies of cellular signaling in cells expressing fluorescently-labeled transgenes. Combined, the methods outlined in this protocol serve as the basis for a flexible experimental approach that can be used to accurately quantify efferocytic activity and interrogate cellular signaling pathways active during efferocytosis.

Efferocytosis, the phagocytic removal of apoptotic cells, is required to maintain homeostasis and is facilitated by receptors and signaling pathways that allow for the recognition, engulfment, and internalization of apoptotic cells. Herein, we present a fluorescence microscopy protocol for the quantification of efferocytosis and the activity of efferocytic signaling pathways.

单细胞荧光显微镜定量 Efferocytosis 的研究

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...

Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a myeloid-biased 39-antibody CyTOF panel to evaluate the hematopoietic system with a focus on the neutrophil-lineage cells by using this protocol. The CyTOF result was analyzed with an open-resource dimensional reduction algorithm, viSNE, and the data was presented to demonstrate the outcome of this protocol. We have discovered new neutrophil-lineage cell populations based on this protocol. This protocol of fresh whole BM preparation may be used for 1), CyTOF analysis to discover unidentified cell populations from whole BM, 2), investigating whole BM defects for patients with blood disorders such as leukemia, 3), assisting optimization of fluorescence-activated flow cytometry protocols that utilize fresh whole BM.

Here, we present a protocol to process fresh bone marrow (BM) isolated from mouse or human for high-dimensional mass cytometry (Cytometry by Time-Of-Flight, CyTOF) analysis of neutrophil-lineage cells.

全骨髓的制备,用于中性粒系细胞的大规模细胞测定分析

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a ...