This work was supported by the Singapore National Research Foundation (grant NRF-RF2010-06 to J.Y.Y.).

1.样品制备<ol><li>穿过与GAL4 3,22驱动线UAS-GCaMP5G 19苍蝇（W 1118，P {20XUAS-IVS-GCaMP5G} attP40）。允许跨在25℃生长。 注:生成与Gal4的或UAS-GCaMP转基因的多个拷贝苍蝇可有助于增强荧光信号的强度。当Gr68a-GAL4驱动程序的控制下表达的UAS-GCaMP转基因（UAS-GCaMP3）的早期版本中表现出非常微弱的荧光强度。 <ol><li>收集和性别新eclosed成蝇分开，在25℃的提高。 注:GCaMP5G花期的基线水平在14-28岁之间的日子过得最佳。按性别隔离苍蝇排除可能影响神经反应社会互动的可能性。 </li></ol></li><li>麻醉下的 CO 2的轻微流一只苍蝇。 </li><li>安装在F立法院通过第一把指甲油一小滴在0.17毫米盖玻片中心的玻璃盖玻片。轻轻地放在透明指甲油的滴在其侧面一只苍蝇，并确保整个降由飞覆盖。 </li><li>执行步骤1.5和1.6解剖立体显微镜，在8倍放大下使用。 </li><li>用湿画笔延长苍蝇的前腿。通过将胶带的两个薄条带在第一和第五跗节（ 图2A）固定在位置上的前腿。这将暴露跗节T2-T4用于成像（ 图2B）。 </li><li>要在长鼻图像的神经元，将磁带薄带过长鼻揭露唇瓣（ 图2C，D）的主席台。 </li><li>周围绘制飞疏水屏障用PAP笔，以防止​​在盖玻片表面流动的解决方案。安装的30分钟内进行测量。 </li></ol> 2.刺激的制备解<ol><li>稀释的亲脂性配体（如CH503，本研究中使用）在PBST溶液:137mM的氯化钠，2.7mM的氯化钾，10毫的 Na 2 HPO 4，1.8毫摩尔KH 2 PO 4，0.1％的Triton X-100（V / V ）; pH值7.4。 <ol><li>使用PBST配体的1毫克/毫升储液。准备使用PBST原液的所有后续稀释液。 注意:根据感兴趣的配体的溶解度，可能需要不同的溶剂以溶解配体。 CH503的在各种溶剂中的溶解性在表1中说明。不同的其它溶剂，PBST没有产生荧光的增加。 </li></ol></li></ol> 3.味觉神经元和图像采集的刺激<ol><li>使用10微升移液器，将10微升PBST搬上跗节。 注意:这一步滋润腿部，防止跗节漂流。 </li><li>使用具有苏相机的光学系统fficient速度和灵敏度，实现高时间分辨率良好的荧光信号。 注:在本研究中，我们采用了旋转盘共聚焦显微镜和SCMOS相机（见材料清单 ）。 </li><li>选择特定的跗段和神经元要被成像。收购三家刺激前期共聚焦Z-栈。 <ol><li>理想的情况下，利用采集设置的足够快，以允许最大的时间分辨率，同时还拍摄了整个细胞的体积。例如收购设置为:200毫秒曝光，分档2×2和6×0.5微米2光学切片，捕捉每2秒。 注意:固定化的腿已被成功地成像为至多10分钟，在30秒的时间间隔，用GCaMP信号的没有明显的漂白效果。对于更长的记录时间，确保腿举行非常牢固就位盖玻片上。 </li><li>优化激光功率为最高的信号 - 噪声整个伊马期间以最小的光漂白实现晋政权。对于这里描述的实验中，在30％的透射率使用491纳米二极管激光器（100毫瓦），用于成像的腿和吸管两者Gr68a和ppk23味觉神经元。用2×2像素合并以允许较短的曝光时间，同时仍实现充分的空间分辨率。 注意:激光功率设置进行了优化，使荧光的变化范围从1.2〜4.5倍的检测。 </li></ol></li><li>移取10微升的刺激解到跗节。对于控制实验中，再添10微升的PBST。立即获取117刺激后的图像。 注:获得刺激后的图像数是基于其中在荧光最大变化达到的时间量（约2分钟）。 <ol><li>关键的一步:虽然在准备吸取溶液，不碰飞枪头，因为这将导致前腿搬出位置。 注:另外，用一个显微精确定位含有刺激接近跗段玻璃毛细管将被成像。递送根据制造商的说明使用的细胞内微注射分配器刺激控制卷。 </li></ol></li></ol> 4.图像处理与分析:响应的定量分析<ol><li>打开使用斐济（一系列共聚焦Z-栈<a href="http://fiji.sc/Fiji" target="_blank">http://fiji.sc/Fiji</a> ）23或ImageJ的（ <a href="http://imagej.nih.gov/ij/" target="_blank">http://imagej.nih.gov/ij/</a> ）24图像分析与处理软件。 </li><li>使每个120的时间点的所有六个ž切片的总和强度投影。 <ol><li>在选择"图像"的"堆栈"选项。此外，选择"Z投影"选项，输入"1"作为起始片，"4"为停止片。对于投影类型，选择R20;总和切片"，从下拉菜单中，选择"所有时间框架"。 </li></ol></li><li>通过使用徒手选择工具绘制一个周围细胞体或神经元投射的边界定义感兴趣区域（ROI）的区域。单击"分析"标签，并选择在"工具"下的"投资回报率管理"选项。 </li><li>通过选择"设置测量"选项，"分析"菜单下并检查盒集成密度，面积量化的投资回报率的荧光，平均和最大灰度值和标签。然后，选择投资回报率经理菜单下的"更多"选项，并进一步选择"多措施"选项。 注意:这将产生与每个所选参数的值的弹出框。 </li><li>检查最大灰度值，以确保没有象素已达到饱和（ 例如 ，65,535为一个16位的相机）。 注:有关实验表明在T要么从根据观察到ΔF/ F中的最大增幅，其中细胞体或神经元的预测，衡量他的论文，反应。 </li><li>在时间点t计算荧光（ΔF/ F）的最大相对变化如下: <img alt="式（1）" src="/files/ftp_upload/53392/53392eq1.jpg" /><ol><li>从每个F（单元）值中减去背景荧光。量化背景荧光，从前腿（或吸管）不含可检测的神经元的面积测量等效尺寸和形状的ROI的集成密度。 </li><li>量化的刺激后的荧光，F（细胞）T，测量在刺激后的时间点"t"的细胞体或过程的ROI的集成密度。 </li><li>量化刺激前期荧光，F（细胞）t = 0 时 ，分析细胞体或工艺的刺激前的图像中的方式相同的刺激后信号。使用3个图像的平均积分密度来确定F（细胞）T = 0。 </li></ol></li></ol>

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The authors declare that there are no competing financial interests.

我们在这里描述在2个不同的感觉器官进行果蝇外周神经元的活钙成像的方法。该钙 -evoked由信息素配体CH503呈剂量依赖性和定量诱导Gr68a神经元GCaMP荧光反应。它也可以辨别不同的神经反应模式，如阶段性和补品响应。 显示相位响应神经元被认为可以快速适应不断刺激。这种类型的响应的已用气味探测25和节奏运动活动图形26的生成有关。相比之?...

动物依靠嗅觉和味觉的信息，以调解的生存和繁衍至关重要的决定。理解暗示如何化学感受检测和由神经系统处理要求感官受体（S）和相应的化学配位体的识别。 果蝇检测了惊人的各种挥发性和非挥发性化合物，并且在其中，研究生理极好模型机制的基本chemosensation。而嗅觉器官感知挥发性分子，味觉器官是专门用于检测低挥发性化合物。在这里，我们提出了直接测量果蝇的味觉器官低挥发性，亲脂性配体神经元的反应的方法。 苍蝇的味觉器官包括前腿，喙，和翅膀。分发的味觉器官的表面上是称为感器毛发状结构，其为s响应ugars，苦味，盐，水和费洛蒙8。该感受器已形态分为刷毛的味道和口感钉9。有关于被分类为长（l型）唇瓣约31味刷毛，短（S型）和中间体（i型）的形态。在&#39;L&#39;和&#39;S&#39;是生理糖回应感器房子4感觉神经元（在S细胞），低盐（L1细胞），高盐和苦味化合物（二级电池）和水（在W单元）10 ，11。的"i"感器房屋2的感觉神经元，其中的一个响应既低盐和糖，而其他响应高盐12。有男性约41的味道感受器和分布在每个前肢女26感器。对于男性和女性中，有21感器上midleg，并在后腿13约22感器。由味道感器上腿包围的味觉神经元还包含classified成L1，L2 W和S的类型。 从单个神经元测量电活动的一种标准方法使用外或细胞内的电极来记录离子流。电极测量允许非模式生物进行研究，例如果蝇 ，蛾，和缺乏神经标记广泛的遗传工具蜜蜂神经功能。然而，尽管电生理方法已被经常使用从果蝇味觉测量活动感器4,13,14，运用这种方法存在一些技术挑战。首先，味道刷毛需要基于形态学和空间位置被识别。经鉴定，电测量可通过感受器的尺寸小的阻碍，限制无障碍由于位置，以及难以应用化学刺激的控制卷味道塞得满满的。另外，感器的刺激可以产生一个以上的信号神经型15。其次，电信号检测可以通过从电子设备的机械振动和噪声产生的背景噪音混淆。三，使用锋利的电极会损坏飞行准备，如果使用不当，14。最后，组装电钻机需要刺激递送，信号记录，和数据分析的专门的电子元件，并且可以是昂贵的。 在D.果蝇的基因编码工具的可用性提供了便利的成像技术，使神经元的小种群的反应进行研究开发。一种这样的方法是使用CaLexA记者16。在此方法中，编码的LexA-VP16转录因子和钙敏感蛋白NFAT基因序列融合在一起。的蛋白磷酸酶钙调磷酸酶的钙活化催化NFAT的去磷酸化，并反过来，facilitaTES其导入到细胞核。在细胞核内时，LexA的结构域结合到其引导绿色荧光蛋白（GFP）报道分子的表达，从而使功能性激活的神经元的持续识别LexAop DNA结合基序。该方法已成功地用于测量以下的活苍蝇暴露于臭剂16具体嗅小球中的触角叶的响应。近日，IR52c味觉受体神经元的生理反应，从使用CaLexA 7活苍蝇测量。然而，对于本研究，这是必要的，以年龄苍蝇为长达6周，以提高GFP转录。同时用抗GFP抗体免疫染色可以用于促进CaLexA信号，这种方法将需要组织固定，从而排除了活细胞成像的可能性。 遗传编码的钙指示剂蛋白GCaMP也被广泛用于研究在一些神经元的反应种17。蛋白质GCaMP前神经元的刺激荧光强度低。刺激的应用程序触发的神经元的动作电位，从而导致钙离子的流入。 GCaMP，势必钙 ，经历构象变化，导致它与亮强度（ 图1）发出荧光。最近开发的单神经元钙成像方法被用于鉴定葡萄糖作为配位体的前腿特定Gr61a味觉神经元18。在这项研究中，从转基因果蝇中Gr61a味觉神经元表达GCaMP解剖前肢上覆盖成像之前琼脂糖层。然而，使用解剖组织可引起GCaMP表达的最终破旧，从而限制了测量时间和检测灵敏度。此外，琼脂糖可以限制灵敏度由于荧光的高背景水平及其光散射特性。 ŧO地址一些弊端，我们描述了使用GCaMP介导的钙成像的记录从前肢和长鼻完整的动物的味觉神经细胞的生理反应。我们表明Gr68a的表达遗传编码GCaMP5G 19至果蝇脂质信息素的生理反应和ppk23神经元，（3 的R，11 Z，19 Z）-3-乙酰氧基11,19-octacosadien -1-醇（CH503）20， 21。神经元的反应是通过量化信息素的刺激过程中GCaMP5G信号的荧光增加测量。在这个协议中，神经元被成像为120秒，这足以区分在各个细胞神经元激活模式中的一个总的持续时间。

<table>
	<tbody>
		<tr>
			<td>Gr68a-Gal4</td>
			<td></td>
			<td></td>
			<td>Gift from H. Amrein (Texas A&#38;M Health Science Center, TX, USA) and J. Carlson (Yale University, CT, USA)</td>
		</tr>
		<tr>
			<td>ppk23-Gal4</td>
			<td></td>
			<td></td>
			<td>Gift from K. Scott (Univ. of California, Berkeley, CA, USA)</td>
		</tr>
		<tr>
			<td>UAS-GCaMP5&#160;</td>
			<td></td>
			<td>42037</td>
			<td>Bloomington Drosophila&#160; Stock Center</td>
		</tr>
		<tr>
			<td>0.17 mm coverslip (Gold-Seal coverslip)</td>
			<td>Electron Microscopy Services</td>
			<td>63790-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Nail polish, &#34;Hard as Nails Clear&#34;&#160;</td>
			<td>Sally Hansen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PAP pen</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>Z377821</td>
			<td></td>
		</tr>
		<tr>
			<td>Paint brush</td>
			<td></td>
			<td></td>
			<td>fine-tipped brush</td>
		</tr>
		<tr>
			<td>Tape&#160;</td>
			<td>Scotch brand</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>13021</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol, lab grade</td>
			<td>Merck</td>
			<td>10094</td>
			<td></td>
		</tr>
		<tr>
			<td>Hexane, HPLC grade</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>H303SK-4</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>472301</td>
			<td></td>
		</tr>
		<tr>
			<td>PBST</td>
			<td></td>
			<td></td>
			<td>Recipe described in the protocol section</td>
		</tr>
		<tr>
			<td>CH503</td>
			<td></td>
			<td></td>
			<td>Synthesis described in Mori et al., 2010</td>
		</tr>
		<tr>
			<td>sCMOS Camera (ORCA Flash4.0)</td>
			<td>Hamamatsu&#160;</td>
			<td>C11578-22U</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope (Ti-Eclipse)</td>
			<td>Nikon</td>
			<td>Ni-E</td>
			<td></td>
		</tr>
		<tr>
			<td>Spinning Disk Scan head&#160;</td>
			<td>Yokogawa</td>
			<td>CSU-X1-A1</td>
			<td></td>
		</tr>
		<tr>
			<td>Aquistion Software (MetaMorph Premier)</td>
			<td>Molecular Devices</td>
			<td>40002</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiji software</td>
			<td>open source</td>
			<td></td>
			<td><a href="http://fiji.sc/Fiji">http://fiji.sc/Fiji</a></td>
		</tr>
	</tbody>
</table>

measuring physiological responses of drosophila sensory neurons to lipid pheromones using live calcium imaging

不同于哺乳动物，昆虫例如果蝇有多个味觉器官。在腿部，长鼻，翅膀和果蝇的产卵的化学感受神经元表达味觉受体1,2，离子通道3-6，和所涉及的检测易失性和非易失性的感官提示的离子型受体7。这些神经元直接联系促味剂，如食品，有毒物质和信息素，因而影响许多复杂的行为如摄食，产卵和配合。电极录音和钙成像已被广泛用于在昆虫量化这些促味剂诱发的神经元的反应。然而，电需要从单一口味感器专用设备和获取测量值可以根据细胞类型，大小和位置在技术上具有挑战性。此外，在果蝇单个神经元分辨率是很困难的，因为味觉感受器后实现本身多于一种类型的化学感受神经元。此处描述的活钙成像法允许测量在活蝇单味觉神经元的响应。这种方法特别适用于成像脂质信息素和其他配体类型的具有在水基溶剂的溶解度低神经元的反应。

使用Gr68a-GAL4或ppk23-GAL4司机GCaMP钙指标进行了基因表达。前脚神经元和非神经细胞支持的不同的种群由每个驱动器（ 图3A-C）标记。在Gr68a-Gal4的观察到亲脂性信息素CH503配体特异性反应和ppk23-Gal4的细胞中表达GCaMP（ 图3D）。在GCaMP5G信号（ΔF/ F）的荧光强度的相对变化，用较高浓度CH503（ 图3E）的增加。有趣的是，CH503的5毫微克?...

Watch this Scientific Journal Video about Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging at JoVE.com

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 ...

measuring-physiological-responses-drosophila-sensory-neurons-to-lipid

Research

JoVE Journal

Immunology and Infection

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

8.5K 次观看.  Temasek Life Sciences Laboratory. 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. 

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

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 ...

科研

免疫与感染

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

测量细菌感染小鼠和免疫反应李斯特菌</em

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection 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 ...

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external environment. The cell can either recover from this insult, which requires active membrane repair processes, or else die depending on the amount of toxin exposure and cell type1. In addition, these toxins induce strong inflammatory responses in infected hosts through activation of immune cells, including macrophages, which produce an array of pro-inflammatory cytokines2. Many Gram positive bacteria produce cholesterol binding toxins which have been shown to contribute to their virulence through largely uncharacterized mechanisms.
Morphologic changes in the plasma membrane of cells exposed to these toxins include their sequestration into cholesterol-enriched surface protrusions, which can be shed into the extracellular space, suggesting an intrinsic cellular defense mechanism3,4. This process occurs on all cells in the absence of metabolic activity, and can be visualized using EM after chemical fixation4. In immune cells such as macrophages that mediate inflammation in response to toxin exposure, induced membrane vesicles are suggested to contain cytokines of the IL-1 family and may be responsible both for shedding toxin and disseminating these pro-inflammatory cytokines5,6,7. A link between IL-1&beta; release and a specific type of cell death, termed pyroptosis has been suggested, as both are caspase-1 dependent processes8. To sort out the complexities of this macrophage response, which includes toxin binding, shedding of membrane vesicles, cytokine release, and potentially cell death, we have developed labeling techniques and fluorescence microscopy methods that allow for real time visualization of toxin-cell interactions, including measurements of dysfunction and death (Figure 1). Use of live cell imaging is necessary due to limitations in other techniques. Biochemical approaches cannot resolve effects occurring in individual cells, while flow cytometry does not offer high resolution, real-time visualization of individual cells. The methods described here can be applied to kinetic analysis of responses induced by other stimuli involving complex phenotypic changes in cells.

Methods for purifying the cholesterol binding toxin streptolysin O from recombinant E. coli and visualization of toxin binding to live eukaryotic cells are described. Localized delivery of toxin induces rapid and complex changes in targeted cells revealing novel aspects of toxin biology.

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 ...

Live Cell Imaging of Alphaherpes Virus Anterograde Transport and Spread

Advances in live cell fluorescence microscopy techniques, as well as the construction of recombinant viral strains that express fluorescent fusion proteins have enabled real-time visualization of transport and spread of alphaherpes virus infection of neurons. The utility of novel fluorescent fusion proteins to viral membrane, tegument, and capsids, in conjunction with live cell imaging, identified viral particle assemblies undergoing transport within axons. Similar tools have been successfully employed for analyses of cell-cell spread of viral particles to quantify the number and diversity of virions transmitted between cells. Importantly, the techniques of live cell imaging of anterograde transport and spread produce a wealth of information including particle transport velocities, distributions of particles, and temporal analyses of protein localization. Alongside classical viral genetic techniques, these methodologies have provided critical insights into important mechanistic questions. In this article we describe in detail the imaging methods that were developed to answer basic questions of alphaherpes virus transport and spread.

Live cell imaging of alphaherpes virus infections enables analysis of the dynamic events of directed transport and intercellular spread. Here, we present methodologies that utilize recombinant viral strains expressing fluorescent fusion proteins to facilitate visualization of viral assemblies during infection of primary neurons.

顺行运输Alphaherpes病毒传播的活细胞成像

Advances in live cell fluorescence microscopy  techniques, as well as the construction of recombinant viral strains that  express fluorescent fusion ...

Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.

Here, we present phenomic approaches for the functional characterization of putative phage genes. Techniques include a developed assay capable of monitoring host anabolic metabolism, the Multi-phenotype Assay Plates (MAPs), in addition to the established method of metabolomics, capable of measuring effects to catabolic metabolism.

噬菌体型组学:生理途径来表征新型病毒蛋白

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage ...

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. 
We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. 
All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

一个<em&gt;体外</em&gt;模型在HIV合并感染的背景下测量免疫应答疟疾

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

Methods to Study Lipid Alterations in Neutrophils and the Subsequent Formation of Neutrophil Extracellular Traps

Lipid analysis performed by high performance thin layer chromatography (HPTLC) is a relatively simple, cost-effective method of analyzing a broad range of lipids. The function of lipids (e.g., in host-pathogen interactions or host entry) has been reported to play a crucial role in cellular processes. Here, we show a method to determine lipid composition, with a focus on the cholesterol level of primary blood-derived neutrophils, by HPTLC in comparison to high performance liquid chromatography (HPLC). The aim was to investigate the role of lipid/cholesterol alterations in the formation of neutrophil extracellular traps (NETs). NET release is known as a host defense mechanism to prevent pathogens from spreading within the host. Therefore, blood-derived human neutrophils were treated with methyl-&#946;-cyclodextrin (M&#946;CD) to induce lipid alterations in the cells. Using HPTLC and HPLC, we have shown that M&#946;CD treatment of the cells leads to lipid alterations associated with a significant reduction in the cholesterol content of the cell. At the same time, M&#946;CD treatment of the neutrophils led to the formation of NETs, as shown by immunofluorescence microscopy. In summary, here we present a detailed method to study lipid alterations in neutrophils and the formation of NETs.

Lipids are known to play an important role in cellular functions. Here, we describe a method to determine the lipid composition of neutrophils, with emphasis on the cholesterol level, by using both HPTLC and HPLC to gain a better understanding of the underlying mechanisms of neutrophil extracellular trap formation.

方法来研究中性粒细胞脂质的改变和中性粒细胞胞外陷阱随后形成

Lipid analysis performed by high performance thin layer chromatography (HPTLC) is a relatively simple, cost-effective method of analyzing a broad range of ...

Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for influenza infections. Microneutralization (MN) assays are often used to measure neutralizing antibody responses in human sera after influenza vaccination or infection. Madine Darby Canine Kidney (MDCK) cells are the commonly used cell substrate for MN assays. However, currently circulating 3C.2a and 3C.3a A(H3N2) influenza viruses have acquired altered receptor binding specificity. The MDCK-SIAT1 cell line with increased &#945;-2,6 sialic galactose moieties on the surface has proven to provide improved infectivity and more faithful replications than conventional MDCK cells for these contemporary A(H3N2) viruses. Here, we describe a MN assay using MDCK-SIAT1 cells that has been optimized to quantify neutralizing antibody titers to these contemporary A(H3N2) viruses. In this protocol, heat inactivated sera containing neutralizing antibodies are first serially diluted, then incubated with 100 TCID50/well of influenza A(H3N2) viruses to allow antibodies in the sera to bind to the viruses. MDCK-SIAT1 cells are then added to the virus-antibody mixture, and incubated for 18 - 20 h at 37 &#176;C, 5% CO2 to allow A(H3N2) viruses to infect MDCK-SIAT1 cells. After overnight incubation, plates are fixed and the amount of virus in each well is quantified by an enzyme-linked immunosorbent assay (ELISA) using anti-influenza A nucleoprotein (NP) monoclonal antibodies. Neutralizing antibody titer is defined as the reciprocal of the highest serum dilution that provides &#8805;50% inhibition of virus infectivity.

Measuring Influenza Neutralizing Antibody Responses to AH3N2 Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Influenza neutralizing antibodies correlate with protection of influenza infections. Microneutralization assays measure neutralizing antibodies in human sera and are often used for influenza human serology. We describe a microneutralization assay using MDCK-SIAT1 cells to measure neutralizing antibody titers to contemporary 3C.2a and 3C.3a A(H3N2) viruses following influenza vaccination or infection.

用 MDCK-SIAT1 细胞 Microneutralization 法测定人血清中中和 (H3N2) 病毒的流感中和抗体反应

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for ...

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Ca2+ is a ubiquitous ion involved in all known cellular processes. While global Ca2+ responses may affect cell fate, local variations in free Ca2+ cytosolic concentrations, linked to release from internal stores or an influx through plasma membrane channels, regulate cortical cell processes. Pathogens that adhere to or invade host cells trigger a reorganization of the actin cytoskeleton underlying the host plasma membrane, which likely affects both global and local Ca2+ signaling. Because these events may occur at low frequencies in a pseudo-stochastic manner over extended kinetics, the analysis of Ca2+ signals induced by pathogens raises major technical challenges that need to be addressed.
Here, we report protocols for the detection of global and local Ca2+ signals upon a Shigella infection of epithelial cells. In these protocols, artefacts linked to a prolonged exposure and photodamage associated with the excitation of Ca2+ fluorescent probes are troubleshot by stringently controlling the acquisition parameters over defined time periods during a Shigella invasion. Procedures are implemented to rigorously analyze the amplitude and frequency of global cytosolic Ca2+ signals during extended infection kinetics using the chemical probe Fluo-4.

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Here, we present protocols to visualize calcium (Ca2+) responses elicited by HeLa cells infected by Shigella. By optimizing the parameters of bacterial infection and imaging with Ca2+ fluorescent probes, atypical global and local Ca2+ signals induced by bacteria over a large range of infection kinetics are characterized.