作者想，感谢理查德休息的SLO质粒和乔纳森·弗兰克斯提供技术援助的慷慨馈赠的厚礼，anthrolysin O，迈克尔Caparon。这项工作是由NIH拨款T32CA82084（PAK），和R01AI072083（RDS）。

1。纯化的链球菌溶血素O（SLO） <ol><li>接种20毫升过夜培养BL21 GOLD细胞含有pBADgIII-SLOhis质粒9到0.5升的LB肉汤，并加入500微升50毫克/毫升氨苄青霉素。在225的转速在37℃下振荡培养直至OD 600，通常为0.6〜1.5小时。在10,000 xg离心5分钟，离心1 ml细菌（考虑T 0），溶解沉淀于140μL1×SDS上样缓冲液/ 1 OD和超声剪切DNA的蛋白质纯度分析。 </li><li>诱导细菌与加入5毫升20％的阿拉伯糖文化，摇动3小时，在室温下在225 rpm的转速下。收集1毫升的细菌，离心机和1×SDS上样缓冲液溶解在上述T 3时间点的纯度分析。 </li><li>收集残留的细菌在离心瓶500毫升，旋转12,000 xg离心12分钟，在4°C（8500转SORVALL GS-3转子）。滗析出上清液，存储沉淀在-80℃过夜或更长的时间，如果需要的话。 </li><li>重悬FR在10毫升冰ozen丸对裂解/洗涤缓冲液（50mM的NaH 2 PO 4，300mM的NaCl，10mM咪唑，pH 8.0）中加入400μl25％的Triton X-100，100微升100毫克/毫升溶菌酶，50补充微升200 1mM苯甲磺酰氟（PMSF）。 50毫升的橡树岭管转移的再悬浮颗粒。 </li><li>用超声波裂解液5次冰用30秒的时间间隔为30秒。在橡树岭管旋转超声裂解为39,000×g离心20分钟在4°C（18,000 rpm的SORVALL SS-34转子）。 </li><li>当溶胞产物的纺纱，洗1毫升用10ml的Ni-NTA琼脂糖裂解/洗涤缓冲液和在1,200 rpm下旋转5分钟，在4℃下（所有进一步的清洗/洗脱在这些条件下使用离心）。吸洗。 </li><li>细菌上清步骤1.5 Ni-NTA琼脂糖，轻轻摇晃2.5小时，在4°C。旋转收集上清。结合10μ4倍SDS上样缓冲液和30μl的上清液，用于纯度分析。洗磁珠4次，裂解/洗涤缓冲液和策中的Tris /盐缓冲液（50mM的Tris，300mM的NaCl，pH8.0）中。 </li><li>洗脱3次，加入1毫升的洗脱缓冲液（250mM咪唑的Tris /盐缓冲液中）和5μl的1M二硫苏糖醇（DTT）的珠粒，在冰上孵育10分钟，纺丝和收集上清液。 SLO是一个非常氧化还原敏感的蛋白，并必须在任何时候都保持在冰上。一旦降低，蛋白质会迅速失去其活性，如果加热至37℃，即使是很短的时间内。 </li><li>洗净500μl的多粘菌素共轭琼脂糖洗脱缓冲液中，自旋为1分钟，在4℃下以10,000 xg和重悬在500μl。要耗尽内毒素，加入200μl珠，每次洗脱和动摇4°C 30分钟。自旋为1分钟，在4℃下，保存上清液以10,000 xg。结合30微升的各洗脱与10μl的4倍的SDS样品缓冲液进行SDS-PAGE分析。 </li><li>测试的纯度通过SDS-PAGE的洗脱。煮沸样品保存在95℃下5分钟，负载10微升10％凝胶上电泳分析。染色GE升用考马斯亮可视化蛋白（ 图2A）。 SLO为69 kDa的。执行一个布雷德福的含量，以确定蛋白浓度（典型的产率是4毫克/毫升首2洗脱，但可能会发生变化的细菌菌株和质粒使用）。 </li><li>测试每个洗脱的溶血活性（见下文）。池洗脱纯度，浓度和溶血性活动是满意的。等分SLO在单次使用的等分试样（通常为5-10微升），在干冰上并存储在-80℃下冻结</li></ol> 2。溶血性贫血含量<ol><li>绵羊红血细胞（红细胞）洗净，在RBC测定缓冲液（0.3％牛血清白蛋白（BSA），2mM的的CaCl 2，10毫摩尔的Hepes，pH值7.4），旋在1,200 rpm离心5分钟，并重新悬浮在2.5％的红细胞10毫升RBC测定缓冲液。 </li><li>加入10μlRBC测定缓冲液/孔在96孔V形底的板置于冰上。串行稀释每洗脱，一式两份。典型的稀释范围为1:1,000到1:512,000。没有毒素，包括3个井3口井10微升2％的Triton X-100的分别作为最小值和最大值的井，。 </li><li>盖板和孵育在37℃下30分钟。在1,200 rpm下离心5分钟。 70微升的上清液转移到平底96孔板中。读A 405。一个单元是50％的红细胞溶解稀释毒素的需要。毒素活性稀释factor/0.01毫升为1个单位。 </li></ol> 3。细胞裂解试验<ol><li>收集细胞，计数，旋转。以2×10 6 /毫升的缓冲液中重悬的R / B（用2mM的RPMI的CaCl 2，0.5％BSA）和20μg/ ml的碘化丙啶。 100μl的细胞加入到96孔V底板。 </li><li>串行稀释SLO 2倍终浓度的缓冲液R / B，加入100μl的毒素或细胞的100微升缓冲R / B。孵育5分钟，37°C。通常，最终的浓度范围从2000 U /毫升至31.25 U /毫升。 </li><li>运行流式细胞仪检测细胞和藻红蛋白（PE）与过滤器收集数据。一个对数移瞬时透CELLS而3个日志位移表明死细胞。计算特定的细胞裂解，减去死细胞的比例（％PI 高 CTL）在控制实验（PI 高 EXP），如下：具体的裂解（％PI 高的 EXP - ％PI 高 CTL） /（100 - ％PI 高 CTL）* 100 </li></ol> 4。微量移液器交货的毒素到细胞在培养皿<ol><li>实验板2×10 5巨噬细胞对胶原蛋白镀膜玻璃底部35毫米盘前一天。 </li><li>打开显微镜和微量注射器。允许加热阶段的时间来预热至37°C显微镜的选择通常取决于对什么是本地可用的。该显微镜需要一个倒置的阶段，一个阶段能够菜肴加热至37℃，激发/发射滤​​波器立方体适合的染料的选择，和空间的物理连接的微量注射器。一个伯特兰透镜是有助益用于显微注射，但不是必需的。驱动显微镜的计算机需要足够的内存来收集和存储数据。 </li><li>标记细胞在37℃下30分钟用染料根据检测，标签可能会做与AM在5微升Fura2 1毫升PBS或2微升钙黄绿素AM在1毫升全媒体。对于Fura2，激发/发射340/510 nm和380/510 nm处收集，和340/380信号的比率确定钙通量。钙黄绿素，激发/发射515分之495，nm，而乙锭二聚体是六百二十零分之五百二十五的纳米和APC是650/660纳米。也可以选择其他的标签。 </li><li>洗涤细胞用PBS和地点在1ml的RPMI补充用2mM CaCl 2的 。安装在显微镜。 </li><li> 4℃下以20,000 xg离心10分钟，稀释毒素和葡聚糖在水中，并且离心通常情况下，1微​​升SLO和4微升10毫克/毫升葡聚糖-555是在6μl水稀释。葡聚糖或其他荧光流体相分子是用来验证该毫微微尖端还没有堵塞，和INJE仙应有的作用。 </li><li>加载毫微微头从后面使用microloader的0.07微升稀释的毒素。 </li><li>毫微微尖装载到微量注射器。调整角度，使喷油器的尖端会坐在中心的细胞与空间各个方向移动。清除的z极限。注射设置应注入0.5秒，在120磅，20磅的反压。降低小费，直到它进入介质。 </li><li>使用伯特兰透镜，中心的前端和后续的尖端被降低，因为它更接近细胞。一旦你失去重心，切换回正常的光学系统。针影子应该是明显的领域。聚焦以上的细胞，并降低针，直到它处于焦点。重点对细胞，并小心地把针相邻的单元格。设置注射的z-限制。展开成像，移动到所需位置的前端，注入，释放毒素在期望的时间点。提高针，移动到一个新区域的细胞注入。针移动到起始位置，以防止任何不想要的针毒素泄漏。 </li></ol>

<ol>
	<li>Walev, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delivery+of+proteins+into+living+cells+by+reversible+membrane+permeabilization+with+streptolysin-O.">Delivery of proteins into living cells by reversible membrane permeabilization with streptolysin-O.</a> Proc. Natl. Acad. Sci. U.S.A. 98, 3185-3190 (2001).</li><li>Walev, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potassium+regulates+IL-1+beta+processing+via+calcium-independent+phospholipase+A2.">Potassium regulates IL-1 beta processing via calcium-independent phospholipase A2.</a> J. Immunol. 164, 5120-5124 (2000).</li><li>Scolding, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesicular+removal+by+oligodendrocytes+of+membrane+attack+complexes+formed+by+activated+complement.">Vesicular removal by oligodendrocytes of membrane attack complexes formed by activated complement.</a> Nature. 339, 620-622 (1989).</li><li>Keyel, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Streptolysin+O+clearance+through+sequestration+into+blebs+that+bud+passively+from+the+plasma+membrane.">Streptolysin O clearance through sequestration into blebs that bud passively from the plasma membrane.</a> J. Cell. Sci. 124, 2414-2423 (2011).</li><li>MacKenzie, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+secretion+of+interleukin-1beta+by+microvesicle+shedding.">Rapid secretion of interleukin-1beta by microvesicle shedding.</a> Immunity. 15, 825-835 (2001).</li><li>Qu, Y., Franchi, L., Nunez, G., Dubyak, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonclassical+IL-1+beta+secretion+stimulated+by+P2X7+receptors+is+dependent+on+inflammasome+activation+and+correlated+with+exosome+release+in+murine+macrophages.">Nonclassical IL-1 beta secretion stimulated by P2X7 receptors is dependent on inflammasome activation and correlated with exosome release in murine macrophages.</a> J. Immunol. 179, 1913-1925 (2007).</li><li>Andrei, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phospholipases+C+and+A2+control+lysosome-mediated+IL-1+beta+secretion:+Implications+for+inflammatory+processes.">Phospholipases C and A2 control lysosome-mediated IL-1 beta secretion: Implications for inflammatory processes.</a> Proc. Natl. Acad. Sci. U.S.A. 101, 9745-9750 (2004).</li><li>Franchi, L., Eigenbrod, T., Munoz-Planillo, R., Nunez, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+inflammasome:+a+caspase-1-activation+platform+that+regulates+immune+responses+and+disease+pathogenesis.">The inflammasome: a caspase-1-activation platform that regulates immune responses and disease pathogenesis.</a> Nat. Immunol. 10, 241-247 (2009).</li><li>Magassa, N., Chandrasekaran, S., Caparon, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Streptococcus+pyogenes+cytolysin-mediated+translocation+does+not+require+pore+formation+by+streptolysin+O.">Streptococcus pyogenes cytolysin-mediated translocation does not require pore formation by streptolysin O.</a> EMBO Rep. 11, 400-405 (2010).</li><li>Pinkney, M., Beachey, E., Kehoe, 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+thiol-activated+toxin+streptolysin+O+does+not+require+a+thiol+group+for+cytolytic+activity.">The thiol-activated toxin streptolysin O does not require a thiol group for cytolytic activity.</a> Infect. Immun. 57, 2553-2558 (1989).</li><li>Watkins, S. C., Salter, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+connectivity+between+immune+cells+mediated+by+tunneling+nanotubules.">Functional connectivity between immune cells mediated by tunneling nanotubules.</a> Immunity. 23, 309-318 (2005).</li><li>McNeil, P. L., Vogel, S. S., Miyake, K., Terasaki, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patching+plasma+membrane+disruptions+with+cytoplasmic+membrane.">Patching plasma membrane disruptions with cytoplasmic membrane.</a> J. Cell. Sci. 113, 1891-1902 (2000).</li><li>Shannon, J. G., Ross, C. L., Koehler, T. M., Rest, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+anthrolysin+O,+the+Bacillus+anthracis+cholesterol-dependent+cytolysin.">Characterization of anthrolysin O, the Bacillus anthracis cholesterol-dependent cytolysin.</a> Infect. Immun. 71, 3183-3189 (2003).</li></ol>

没有利益冲突的声明。

这里描述的技术允许检查细菌毒素的免疫细胞的反应。最关键的步骤是处理和剂量的毒素。毒素活性可以是非常可变之间，甚至在不同的等分试样的相同的制备，由于其脆弱性。因此，有必要对引用单元格的行或红细胞的毒素测试各等份，或使用毒素梯度的。毒素梯度，交付微量移液器，让毒素引起的活动时必须遵守的实时全方位的，但不适合本身的生化分析。 微管交货的?...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td> Ni-NTA琼脂糖</td><td> Qiagen公司</td><td> 30210 </td><td></td></tr><tr><td>多粘菌素琼脂糖</td><td>西格玛</td><td> P1411-5ML </td><td></td></tr><tr><td>泽巴脱盐旋转山坳</td><td>费舍尔</td><td> PI-89891 </td><td></td></tr><tr><td>绵羊红细胞</td><td>费舍尔</td><td> 50-415-688 </td><td></td></tr><tr><td> pBADgIII-SLO </td><td> N / A </td><td> N / A </td><td>见文献9 </td></tr><tr><td> Cy5的monoreactive染料</td><td> GE医疗集团</td><td> PA25001 </td><td></td></tr><tr><td> Fura2-AM </td><td> Life Technologies公司</td><td> F1221 </td><td></td></tr><tr><td>钙黄绿素AM / EThidium二聚体</td><td> Life Technologies公司</td><td> L3224 </td><td></td></tr><tr><td>抗CD11c-APC </td><td> BD Biosciences公司</td><td> 550261 </td><td></td></tr><tr><td>胶原涂层的玻璃底培养皿</td><td> Mattek </td><td> P35GCol-1.5-10-C </td><td></td></tr><tr><td>毫微微尖II </td><td>费舍尔</td><td> E5242957000 </td><td></td></tr><tr><td> Microloader </td><td>费舍尔</td><td> E5242956003 </td><td></td></tr><tr><td>右旋糖酐Alexa的555 </td><td> Life Technologies公司</td><td> D34679 </td><td></td></tr><tr><td> Injectman NI 2 </td><td> Eppendorf公司</td><td> 920000029 </td><td></td></tr><tr><td> FemtoJet </td><td> Eppendorf公司</td><td> 5247 000.013 </td><td></td></tr><tr><td></td><td></td><td></td><td> 表1中。列表和源的具体试剂和equipm耳鼻喉科需要。在该协议中使用的特殊设备和试剂，以及公司和产品目录号上市。 </td></tr><tr><td></td><td></td><td></td><td><table border="1"><tbody><tr><td> 缓冲区 </td><td> 组成 </td><td> 步骤使用 </td></tr><tr><td>裂解/清洗</td><td>的50mM的NaH 2 PO 4的 300mM的NaCl 的10mM咪唑，pH 8.0 </td><td> 1.4 </td></tr><tr><td>三/盐</td><td>的50mM Tris，pH值8.0 300mM的NaCl </td><td> 1.7 </td></tr><tr><td>洗脱缓冲液</td><td>的50mM Tris，pH值8.0 300mM的NaCl 250 mM咪唑</td><td> 1.8 </td></tr><tr><td> RBC分析缓冲液</td><td> 0.3％BSA 2毫米氯化钙 的10mM HEPES，pH值7.4 </td><td> 2.1 </td></tr><tr><td>缓冲R / B </td><td>的RPMI细胞培养基 2毫米氯化钙 0.5％BSA的</td><tD&gt; 3.1 </td></tr></tbody></table></td></tr><tr><td></td><td></td><td></td><td> 表2列出在本协议中所使用的缓冲区。使用的缓冲液，它们的组合物和它们在协议中使用的第一个步骤中列出。 </td></tr></tbody></table>

visualization of bacterial toxin induced responses using live cell fluorescence microscopy

细菌毒素结合到膜中的胆固醇，细胞内容物的泄漏，和来自外部环境的材料涌入允许形成细孔。从这个侮辱，这需要积极的膜修复过程的细胞可以恢复，否则死取决于毒素的暴露量和细胞类型1。此外，这些毒素诱导强烈的炎症反应，在受感染的宿主中，通过激活免疫细胞，包括巨噬细胞，从而产生的促炎性细胞因子的阵列2。许多革兰氏阳性细菌产生的胆固醇结合的毒素，这已被证明有助于其毒力，很大程度上是未知的机制。 这些毒素的细胞暴露在质膜的形态学改变包括其螯合成富含胆固醇的表面突起，它可以脱落到细胞外空间，提示的特性的细胞防御机制3,4。此过程发生的代谢活动的情况下，在所有细胞中，可以可视化后使用EM化学固定4。在免疫细胞如巨噬细胞介导炎症的毒素暴露，引起膜泡的建议包含细胞因子IL-1家族，可能是负责脱落毒素和传播这些促炎性细胞因子5,6,7。 IL-1β的释放之间的链接，以及一种特定类型的细胞死亡，称为pyroptosis已建议，因为二者都是caspase-1的依赖过程8。进行排序，满分该巨噬细胞反应的复杂性，其中包括毒素结合，脱落的膜性囊泡，细胞因子的释放，和潜在的细胞死亡，我们已经开发了标记技术和荧光显微镜观察的方法，使毒素的细胞间的相互作用的实时可视化的，包括测量功能障碍和死亡（ 图1）。使用活细胞成像是必要的，因为其他技术的限制。生化方法不能解决发生在单个细胞的效应，而流式细胞仪没有提供高分辨率，单个细胞的实时可视化。这里描述的方法可以适用于由其他涉及复杂的细胞的表型变化的刺激诱导的反应的动力学分析。

通常为10 7 -10 8 U / ml的SLO可以得到4毫克/毫升的蛋白浓度。毒素细胞裂解所需的量的细胞类型而变化，但通常为125-500 U / ml的SLO（ 图2B）。如巨噬细胞的细胞类型可以更耐（4000 U /毫升），但其他人（特别是T细胞系）更为敏感。这些敏感性与市售SLO。毒素活性降低大约2倍的与各冻结 - 解冻，因此溶血检测与毒素每批或解冻需要验证毒素活性。毒素是在的情况下，含胆固...

Watch this Scientific Journal Video about Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy at JoVE.com

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

方法从重组净化胆固醇结合毒素链球菌溶血素O<em&gt; E.大肠杆菌</em&gt;和可视化的生活真核细胞毒素结合进行了阐述。本地化的交付毒素诱导靶细胞，揭示新的毒素生物学方面的快速而复杂的变化。

细菌毒素引起的响应，用活细胞荧光显微镜的可视化

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external ...

visualization-bacterial-toxin-induced-responses-using-live-cell

Research

JoVE Journal

Immunology and Infection

11.7K 次观看.  University of Pittsburgh School of Medicine. 方法从重组净化胆固醇结合毒素链球菌溶血素O E.大肠杆菌和可视化的生活真核细胞毒素结合进行了阐述。本地化的交付毒素诱导靶细胞，揭示新的毒素生物学方面的快速而复杂的变化。

视频: Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Visualizing Cell-to-cell Transfer of HIV using Fluorescent Clones of HIV and Live Confocal Microscopy

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using fluorescence video microscopy. To track the movements of the human immunodeficiency virus core protein during cell-to-cell transmission of human immunodeficiency virus, we have GFP-tagged the Gag protein in the context of an infectious molecular clone of HIV, called HIV Gag-iGFP. We study this viral clone using video confocal microscopy. In the following visualized experiment, we transfect a human T cell line with HIV Gag-iGFP, and we use fluorescently labeled uninfected CD4+ T cells to serve as target cells for the virus. Using the different fluorescent labels we can readily follow viral production and transport across intercellular structures called virological synapses. Simple gas permeable imaging chambers allow us to observe synapses with live confocal microscopy from minutes to days. These approaches can be used to track viral proteins as they move in from one cell to the next.

This visualized experiment is a guide for utilizing a fluorescent molecular clone of HIV for live confocal imaging experiments.

可视化的细胞与细胞转移的艾滋病毒，艾滋病毒和现场共聚焦显微镜的荧光克隆

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using ...

科研

免疫与感染

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of guard cells, which are able to close the stomatal pore in response to a number of external factors including light intensity, carbon dioxide concentration, and relative humidity (RH). The stomatal pore is also the main route for pathogen entry into leaves, a crucial step for disease development. Recent studies have unveiled that closure of the pore is effective in minimizing bacterial disease development in Arabidopsis plants; an integral part of plant innate immunity. Previously, we have used epidermal peels to assess stomatal response to live bacteria (Melotto et al. 2006); however maintaining favorable environmental conditions for both plant epidermal peels and bacterial cells has been challenging. Leaf epidermis can be kept alive and healthy with MES buffer (10 mM KCl, 25 mM MES-KOH, pH 6.15) for electrophysiological experiments of guard cells. However, this buffer is not appropriate for obtaining bacterial suspension. On the other hand, bacterial cells can be kept alive in water which is not proper to maintain epidermal peels for long period of times. When an epidermal peel floats on water, the cells in the peel that are exposed to air dry within 4 hours limiting the timing to conduct the experiment. An ideal method for assessing the effect of a particular stimulus on guard cells should present minimal interference to stomatal physiology and to the natural environment of the plant as much as possible. We, therefore, developed a new method to assess stomatal response to live bacteria in which leaf wounding and manipulation is greatly minimized aiming to provide an easily reproducible and reliable stomatal assay. The protocol is based on staining of intact leaf with propidium iodide (PI), incubation of staining leaf with bacterial suspension, and observation of leaves under laser scanning confocal microscope. Finally, this method allows for the observation of the same live leaf sample over extended periods of time using conditions that closely mimic the natural conditions under which plants are attacked by pathogens.

We have developed a simple and reproducible protocol to access stomatal response to live bacteria. This method minimizes wounding and manipulation of the leaf as compared to the use of epidermal peels reported previously.

评估气孔反应活细菌细胞，采用全叶成像

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of ...

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 ...

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 ...

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of phagocytes to the site where pathogens are located; (ii) recognition of pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs); (iii) engulfment of microorganisms bound to the phagocyte cell membrane, and (iv) processing of engulfed cells within maturing phagosomes and digestion of the ingested particle. Studies that assess phagocytosis in its entirety are informative1, 2, 3, 4, 5 but are limited in that they do not normally break the process down into migration, engulfment and phagosome maturation, which may be affected differentially. Furthermore, such studies assess uptake as a single event, rather than as a continuous dynamic process. We have recently developed advanced live-cell imaging technologies, and have combined these with genetic functional analysis of both pathogen and host cells to create a cross-disciplinary platform for the analysis of innate immune cell function and fungal pathogenesis. These studies have revealed novel aspects of phagocytosis that could only be observed using systematic temporal analysis of the molecular and cellular interactions between human phagocytes and fungal pathogens and infectious microorganisms more generally. For example, we have begun to define the following: (a) the components of the cell surface required for each stage of the process of recognition, engulfment and killing of fungal cells1, 6, 7, 8; (b) how surface geometry influences the efficiency of macrophage uptake and killing of yeast and hyphal cells7; and (c) how engulfment leads to alteration of the cell cycle and behavior of macrophages 9, 10.
In contrast to single time point snapshots, live-cell video microscopy enables a wide variety of host cells and pathogens to be studied as continuous sequences over lengthy time periods, providing spatial and temporal information on a broad range of dynamic processes, including cell migration, replication and vesicular trafficking. Here we describe in detail how to prepare host and fungal cells, and to conduct the video microscopy experiments. These methods can provide a user-guide for future studies with other phagocytes and microorganisms.

We describe methods for live-cell video microscopy of Candida albicans phagocytosis by macrophages. These methods enable stage-specific analysis of macrophage migration, recognition, engulfment and phagosome maturation and reveal novel aspects of phagocytosis.

活细胞视频显微镜病病原菌的吞噬作用

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of ...

Application of Long-term cultured Interferon-&#947; Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is controlled up to 95 % of the T cells generated during the expansion phase are eliminated (i.e., contraction phase) and memory T cells remain, sometimes for a lifetime. In humans, two functionally distinct subsets of memory T cells have been described based on the expression of lymph node homing receptors. Central memory T cells express C-C chemokine receptor 7 and CD45RO and are mainly located in T-cell areas of secondary lymphoid organs. Effector memory T cells express CD45RO, lack CCR7 and display receptors associated with lymphocyte homing to peripheral or inflamed tissues. Effector T cells do not express either CCR7 or CD45RO but upon encounter with antigen produce effector cytokines, such as interferon-&#947;. Interferon-&#947; release assays are used for the diagnosis of bovine and human tuberculosis and detect primarily effector and effector memory T cell responses. Central memory T cell responses by CD4+ T cells to vaccination, on the other hand, may be used to predict vaccine efficacy, as demonstrated with simian immunodeficiency virus infection of non-human primates, tuberculosis in mice, and malaria in humans. Several studies with mice and humans as well as unpublished data on cattle, have demonstrated that interferon-&#947; ELISPOT assays measure central memory T cell responses. With this assay, peripheral blood mononuclear cells are cultured in decreasing concentration of antigen for 10 to 14 days (long-term culture), allowing effector responses to peak and wane; facilitating central memory T cells to differentiate and expand within the culture.

Application of Long-term cultured Interferon-&amp;#947; Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

Long-term cultured interferon-&#947; enzyme-linked immunospot assay is used as a measure of central memory responses and correlates with protective anti-mycobacterial vaccine responses. With this assay, peripheral blood mononuclear cells are stimulated with mycobacterial antigens and interleukin-2 for 14 days, enabling differentiation and expansion of central memory T cells.

长期培养的干扰素γ酶联免疫检测方法的评估效应和记忆T细胞应答的牛应用

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

生成和多表型高内涵筛选<em&gt;贝氏柯克斯体</em&gt;转座子突变体

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila express gustatory receptors1,2, ion channels3-6, and ionotropic receptors7 that are involved in the detection of volatile and non-volatile sensory cues. These neurons directly contact tastants such as food, noxious substances and pheromones and therefore influence many complex behaviors such as feeding, egg-laying and mating. Electrode recordings and calcium imaging have been widely used in insects to quantify the neuronal responses evoked by these tastants. However, electrophysiology requires specialized equipment and obtaining measurements from a single taste sensillum can be technically challenging depending on the cell-type, size, and position. In addition, single neuron resolution in Drosophila can be difficult to achieve since taste sensilla house more than one type of chemosensory neuron. The live calcium imaging method described here allows responses of single gustatory neurons in live flies to be measured. This method is especially suitable for imaging neuronal responses to lipid pheromones and other ligand types that have low solubility in water-based solvents.

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

The forelegs and proboscis of Drosophila contain a rich repertoire of gustatory sensory neurons. Here, we present a method using calcium imaging to measure physiological responses from sensory neurons in the foreleg and proboscis of live flies upon exogenous application of a gustatory pheromone.

测量的生理反应<em&gt;果蝇</em&gt;感觉神经元脂质费洛蒙使用实时钙成像

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila ...

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 ...

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' pathogens (Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis, and Rickettsia spp.) gain access to the infected cell cytosol by physically and enzymatically degrading the primary vacuolar membrane. Once in the cytosol, these pathogens both proliferate as well as generate sufficient mechanical forces to penetrate the plasma membrane of the host cell in order to infect new cells. Here, we show how this terminal step of the cellular infection cycle of L. monocytogenes (Lm) can be quantified by both colony-forming unit assays and flow cytometry and give examples of how both pathogen- and host-encoded factors impact this process. We also show a close correspondence of Lm infection dynamics of cultured cells infected in vitro and those of hepatic cells derived from mice infected in vivo. These function-based assays are relatively simple and can be readily scaled up for discovery-based high-throughput screens for modulators of eukaryotic cell function.

We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.