这项研究是由美国农业部农业研究服务支持。本出版物中的商品名称或商业产品只是提到提供具体信息之目的，并不由农业部美国农业部暗示推荐或认可。美国农业部是一个平等机会提供者和雇主。

1.细菌菌落外周边缘<ol><li> 十，成长fastidiosa（Xf为 ）蒂梅丘拉野生型22，pilG缺失突变Xf为 ΔpliG（使用先前描述删除策略23），以及其互补XfΔpliG-C（使用先前描述基于染色体遗传互补策略24）上的 PD2培养基琼脂板25在28℃下5-7天。 </li><li>高压釜玻璃纸（1×1 cm 2）的水在121℃（249°F）的15分钟。拿起一块玻璃纸，通过在一个空的培养皿接触玻璃纸的一个角落里沥干水，仔细地打好玻璃纸在琼脂表面和空气干燥的15％。 </li><li>拿起个别X. fastidiosa菌落用无菌牙签圆润和现货细胞无菌到在琼脂平板覆盖在琼脂表面的15％的玻璃纸杀菌片。孵育所述板■在28°C 2-3天。 </li><li>检查使用具有2X物镜解剖显微镜和10X目镜殖民地的边缘形态。照片周围的殖民地外围边缘。 </li></ol> 2.显微镜和微流控流动室<ol><li>先前描述5,18使用类似于光刻过程制造微流体装置。使用标准光刻方法26设计与电脑辅助设计软件的主硅片上四个并行通道。 </li><li>与聚二甲基硅氧烷（PDMS）创建一个从硅片主微流控室。倒未聚合的PDMS在硅片主，并在60℃下固化它为1小时。剥离从晶片主与PDMS复制品并修剪PDMS复制品用刀片成22毫米×40mm的作为玻璃盖玻片的大小相同。 </li><li>露出PDMS复制品，在玻璃盖玻片（22×40mm的2），以及一个microscOPE幻灯片（为50 x 75 平方毫米）空气等离子体在30W 2分钟27。夹层玻璃盖玻片，玻璃显微镜打造微FL uidic室之间的PDMS身体。 </li><li>在图案化的通道的每一端钻透PDMS中的孔（5.5毫米直径）。切硅橡胶管插入长12-20厘米。插入硅橡皮管的一端（外径5.1毫米，内径2.1毫米，0.8毫米壁）插入PDMS复制品的信道的各开口端部，并在60℃下用未聚合的PDMS密封它为1小时。 </li><li>管子的另一端连接到塑料鲁尔连接器的倒刺末端。包组装微流体腔室与铝箔和高压灭菌他们20分钟。 </li><li> 十，收集细菌细胞fastidiosa野生型，突变体Xf为 ΔpliG，互补XfΔpliG-C通过刮，用一次性接种从PD2中板循环。调整细胞密度到0.05在PD2肉汤600纳米的光密度如前面23所描述。收集细菌细胞溶液到1毫升气密注射器。 </li><li>安装在倒置显微镜阶段微流体装置。入口管连接到包含PD2肉汤中的5毫升注射器气密性。适应中的5毫升注射器气密用注射器泵。 </li><li>出口管连接到废物储存器。保持0.2微升min的介质流速-1 30分钟以稳定该系统。 </li><li>侧入口管连接到含有细菌细胞溶液1毫升气密注射器。冲洗通过橡胶管的细菌细胞溶液，直到达到信道。以冲洗从室图像捕获之前未结合的细胞保持0.2微升min的介质流速-1另外的30分钟。 </li><li>安装在显微镜来控制光的场调整部分在显微镜下快门。活门连接到快门共ntrol系统和闸门控制系统连接到计算机。 </li><li>安装一台数码相机到显微镜的视频端口，并将其连接到电脑。运行时间推移录音软件，从菜单中选择了“快门”功能，并自动识别软件安装快门为默认建立与软件的快门连接。 </li><li>从时间推移刻录软件的菜单中选择“数码相机”功能可以自动识别数码相机作为软件默认的捕获设备，并建立与数码相机相关的软件通信。 </li><li>定位在使用20X相衬光学通道之一的细菌细胞，然后切换到之前的图像捕获的40X物镜。 </li><li>运行时间推移录音软件，使用从菜单中默认参数，以获得从显微镜图像选择“图像采集”功能。接下来，打开“采集延时”功能，时间间隔设置为30秒5,18,28为根据，以观察X的运动性抽动实验需要6-24小时的时间fastidiosa 5,18,28。点击“确定”，启动延时录制。点击“堆栈功能”，从菜单中选择“另存为”，以完成录音后，堆在目标文件夹中的图像在计算机上。 </li><li>对于多个通道，从第一通道捕获时间推移的图像，每隔30秒6小时。移动显微镜的物镜到下一个通道来定位目标细胞。如上所述，如果实验设置为利用四个通道捕捉图像中的每一个顺序地四个通道重复时间推移功能。继续只要连续三天的时间推移图像采集。所有实验均在室温（23±2℃）下进行。 </li><li>编译时间推移图像成使用时间推移录制的图像处理软件的视频文件。运行时间推移录音软件，单击“堆栈功能”，从菜单，并选择“打开堆叠功能”，从计算机打开堆放的文件。 </li><li>从堆栈模块启动“制作电影”功能，选择所有图像并选择“AVI”格式输出。点击“另存为”的视频文件存储在计算机上的目标文件夹。 </li><li>选择在计算机上从目标文件夹编译电影并播放它们。然后，通过细菌细胞的抽搐运动活动中所生成的视频文件生成的可视化观察单个细胞的运动。 </li></ol>

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作者宣称，他们没有竞争的经济利益。

在这项研究中，我们的特点X的运动行为fastidiosa PilG突变Xf为 ΔpilG和新设计的多个并行的纳米通道微FL uidic室互补XfΔpilG-Ç株。新设计的微FL uidic腔室可以具有100微米的纳米通道多达四个平行腔室宽度相比仅与单个50微米宽的通道18早期的设计。改进的更宽纳米通道便于引入细菌细胞与媒体的流动。此外，该微流体室1）简单的构建和组装; 2）相对便宜;?...

叶缘焦枯病菌是革兰氏阴性非鞭 ​​毛，病原菌引起许多经济上重要的作物疾病，包括皮尔斯在葡萄病（ 葡萄属）1,2，3条。这种细菌是有限的导水木质部船只。葡萄感染引起木质部导管和水分胁迫的结果和营养不足3堵塞。成功定植取决于细菌的从感染的初始位点移动到植物3的其余部分的能力。抽搐蠕动是鞭毛无关细菌移动的装置通过扩展，附件，和极性IV型菌毛4已特征在于X的缩回fastidiosa 5,6,7。 抽搐运动已由激光镊子和原子力显微镜（AFM）8,9,10观察。使用这些技术，叔由N型菌毛IV产生的魔力motilities 淋球菌和P.由FL uorescently标签菌毛和显微镜捕捉他们的动作绿脓杆菌进行了表征。虽然这两种方法有详细的个体细菌的粘合力，该程序是复杂和耗时9,10。微FL uidic室，观察单个细胞的长途迁徙，以及细菌细胞5,6小聚集。这些室被设计成与隔时图像记录系统11,12,13,14一体的板一个微制造纳米通道。微FL uidic室装置用于研究细菌的运动行为和细胞 - 细胞相互作用提供几个优点：（i）其提供了具有多个信道功能的集成平台; （ii）其可以检查运动和单细胞的细菌的纳米级特征的聚合; （三）它允许直接米细菌细胞和时间推移分析icroscopic图像记录，（ⅳ）它提供了一种在微环境的细菌的个体和/或群体的长期的空间和时间的观测; （五）培养基中的信道的流量可以精确控制和培养基（ⅵ）仅有一个非常小的体积（1ml）中，需要对每个实验。 最近，微FL uidic溢流系统已被用于研究在各种微环境14,15,16细菌细胞的行为。的密合性和E的表面附着大肠杆菌 15，X。 fastidiosa 16和敏捷食酸citrulli 14到玻璃表面用微FL uidic室评估。由敏捷食citrulli的IV型菌毛介导的聚集和生物膜的形成进行了分析14。另外，A的运动citrulli下溢流Ç观察onditions表明，IV型菌毛可能在殖民化中发挥重要作用，传播的A. citrulli在木质部导管在边溢流条件。 铜绿假单胞菌和X的抽搐motilities fastidiosa细胞在微加工流动室5,6,17针对流体当前成功观察。 IV型菌毛短少X的pilB和pilQ突变fastidiosa被发现深刻地改变微FL uidic装置5,6,18的溢流条件下抽搐运动的速度。在微FL uidic器件细菌粘附和运动进行的研究表明，微FL uidic室是特别适合于在体外分析菌毛介导的细菌的抽搐运动和迁移。这些结果可以解释这有利于细胞间的连接，聚集和殖民内的抽动介导的迁移机制主机，最终导致全身感染。 X的弼热电联产操纵子fastidiosa包含pilG，霹雳，pilJ，丸剂，chpB和CHPC其中编码信号转导通路20。跨膜化学感受器结合化学刺激在周质结构域和在其细胞质部分以最终控制细菌抽搐蠕动激活信号级联。在X的弼CHP操纵子fastidiosa，一个磷酸化蛋白班车是PilG同源到崔泰源。在E.大肠杆菌和P.假单胞菌，崔泰源是在与该鞭毛马达蛋白19，21交互趋化系统的响应调节。虽然弼CHP操纵子在X的贡献对毒力fastidiosa最近检查20，pilG在趋操纵子的作用，响应于环境信号及X的调节/马达IV型菌毛fastidiosa是UNC李尔。为了阐明X的抽动运动的活动趋调节pilG的洞察力fastidiosa，微FL uidic室被用来评估X的抽搐蠕动fastidiosa。 十fastidiosa的pilG的特征在于，通过比较一个缺失突变体 Xf为 ΔpliG，互补应变XfΔpliG-C及其野生型在体外 的表型 。结果突出pilG在X的抽动运动中的作用fastidiosa。

<table>
	<tbody>
		<tr>
			<td>Biology materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>X. fastidiosa (Xf) Temecula wild type</td>
			<td></td>
			<td></td>
			<td>Costa, H. S., et al., 2004 22</td>
		</tr>
		<tr>
			<td>pilG deletion mutant Xf&#916;pliG</td>
			<td></td>
			<td></td>
			<td>Shi, X. Y., et al., 2007 26</td>
		</tr>
		<tr>
			<td>pilG complementary strain Xf&#916;pliG-C&#160;</td>
			<td></td>
			<td></td>
			<td>Davis, M. J., wt al. 1998 23</td>
		</tr>
		<tr>
			<td>Physical materials and equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable inoculating loops</td>
			<td>VWR international, Radnor, PA</td>
			<td>#22-363-607</td>
			<td>quantitative procedures such as bacterial collection</td>
		</tr>
		<tr>
			<td>Polydimethylsiloxane (PDMS)</td>
			<td>Dow Corning Corporation</td>
			<td>#0002709226</td>
			<td>Sylgard 184 silicone Elastomeric Kits</td>
		</tr>
		<tr>
			<td>AmScope MD2000 digital camera</td>
			<td>AmScope, Irvine, CA</td>
			<td>SE305R-AZ-E</td>
			<td>Image, video recording and measurement&#160;</td>
		</tr>
		<tr>
			<td>Tubes line</td>
			<td>Edgewood, NY</td>
			<td>#T4300</td>
			<td>Connected to the syringe and microfluidic chamber</td>
		</tr>
		<tr>
			<td>Plastic luer connectors</td>
			<td>Edgewood, NY</td>
			<td></td>
			<td>Connected to the syringe and microfluidic chamber</td>
		</tr>
		<tr>
			<td>Syringe pumps</td>
			<td>Pico Plus, Harvard Apparatus, MA</td>
			<td>#702209</td>
			<td>The flow rate can be adjusted while the pump is running.</td>
		</tr>
		<tr>
			<td>Syringes</td>
			<td>Gastight, Hemilton Company, Reno, NV</td>
			<td>#1005</td>
			<td>Provide the flowing broth</td>
		</tr>
		<tr>
			<td>Inverted Olympus IMT-2 microscope</td>
			<td>Olympus</td>
			<td>IMT-2 FLuoro PHase</td>
			<td>Image observation and recording</td>
		</tr>
		<tr>
			<td>SPOT-RT digital camera</td>
			<td>Diagnostic Instruments, Inc., MI</td>
			<td>RT230</td>
			<td>Image, video recording and measurement</td>
		</tr>
		<tr>
			<td>Microscope Shutter</td>
			<td>The UNIBLITZ, US</td>
			<td>#LS2T2</td>
			<td>Control camera&#8217;s exposure time</td>
		</tr>
		<tr>
			<td>Microscope Shutter Control system</td>
			<td>The UNIBLITZ, US</td>
			<td>VCM-D1</td>
			<td>VCM-D1 Single Channel CE/UL/CSA Approved Shutter Driver</td>
		</tr>
		<tr>
			<td>MetaMorph Image software</td>
			<td>Universal Imaging Corp., PA</td>
			<td></td>
			<td>Real-time super-resolution image processing&#160;</td>
		</tr>
	</tbody>
</table>

visualization of twitching motility and characterization of the role of the pilg in xylella fastidiosa

叶缘焦枯病菌是革兰氏阴性非鞭 ​​毛的细菌引起的若干植物的经济上重要的疾病。抽搐蠕动提供X. fastidiosa长距离内厂运行和殖民化，手段在十朝贡献致病fastidiosa，X的运动性抽动fastidiosa是由IV型菌毛操作。 叶缘焦枯病菌的IV型菌毛由pilG，在弼CHP操纵子编码的蛋白质趋化性调节器所涉及的信号转导通路调节。为了阐明pilG的角色X的抽动运动fastidiosa，一个pilG缺陷型突变体Xf为 ΔpilG及其互补株XfΔpilG-含有C本地pilG被开发。与时间推移图像记录系统集成的微流控室，观察抽搐蠕动在XfΔpILG，XfΔpilG-C及其野生型菌株。 利用这种记录系统中，其允许聚合的长期的空间和时间的观察，个体细胞，并通过抽动蠕动细菌种群的迁移。X. fastidiosa野生型和互补XfΔpilG-C株显示，微流体流室直接观察到典型的抽动运动特征，而突变XfΔpliG展出抽搐缺陷表型。这项研究表明，pilG有助于X的运动性抽动fastidiosa。微流体流动室被用作用于观察抽动运动的装置。

表示IV型菌毛介导的抽搐运动的周菌落边缘的情况下，在X的菌落观察fastidiosa野生型和互补Xf为 ΔpliG-C菌株（ 图1）。突变XfΔpliG，然而，并没有表现出在菌落周围的周边的边缘（ 图1）。在纳米微流体流动腔室的细菌细胞的延时成像显示，在野生型X的观察抽搐蠕动fastidiosa和互补XfΔpliG-C（补充V1?...

Watch this Scientific Journal Video about Visualization of Twitching Motility and Characterization of the Role of the PilG in Xylella fastidiosa at JoVE.com

Visualization of Twitching Motility and Characterization of the Role of the PilG in Xylella fastidiosa

在这项研究中，纳米微流体流动室被用于可视化和功能上表征叶缘焦枯病菌，导致皮尔斯病在葡萄细菌的抽搐蠕动。

抽动运动功能的可视化和表征的角色的<em&gt; PilG</em&gt;在<em&gt;叶缘焦枯病菌</em

Xylella fastidiosa is a Gram-negative non-flagellated bacterium that causes a number of economically important diseases of plants. The twitching motility ...

The over goal of this protocol is to functionally characterize the genes responsbile for the twitching motility of bacteria using a nano microfluidic flow chamber integrated with a time-lapse image recording system.This method can help answer key questions about a bacteria phenotype and associated gene function, such as the twitching motility of bacteria.The main advantage of this technique is that it provides a special and a tempered observation for aggregation migration of individual cells and populations.Demonstrating this procedure will be Dr.Xiangyang Shi, a post-doc from my laboratory.Using a standardized deletion strategy, grow Temecula wild-type Xylella fastidiosa, grow pilG deletion mutants, and grow the complimentary strain all on PD2 medium agar plates.Incubate the bacteria at 28

visualization-twitching-motility-characterization-role-pilg-xylella

Research

JoVE Journal

Immunology and Infection

Visualization of Twitching Motility and Characterization of the Role of the PilG in Xylella fastidiosa

10.6K 次观看.  University of California, Davis. The over goal of this protocol is to functionally characterize the genes responsbile for the twitching motility of bacteria using a nano microfluidic flow chamber integrated with a time-lapse image recording system.This method can help answer key questions about a bacteria phenotype and associated gene function, such as the twitching motility of bacteria.The main advantage of this technique is that it provides a special and a tempered observation for aggregation migration of individual cells ...

视频: Visualization of Twitching Motility and Characterization of the Role of the PilG in Xylella fastidiosa

Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue (NALT) in Mouse Responses to Vaccines

The nasopharyngeal-associated lymphoreticular tissues (NALT) found in humans, rodents, and other mammals, contribute to immunity in the nasal sinuses1-3. The NALT are two parallel bell-shaped structures located in the nasal passages above the hard palate, and are usually considered to be secondary components of the mucosal-associated lymphoid system4-6. Located within the NALT are discrete compartments of B and T lymphocytes interspersed with antigen-presenting dendritic cells4,7,8. These cells are surrounded by an epithelial cell layer intercalated with M-cells that are responsible for antigen retrieval from the mucosal surfaces of the air passages9,10. Naive lymphocytes circulating through the NALT are poised to respond to first encounters with respiratory pathogens7. While NALT disappear in humans by the age of two years, the Waldeyer's Ring and similarly structured lymphatic organs continue to persist throughout life6. In contrast to humans, mice retain NALT throughout life, thus providing a convenient animal model for the study of immune responses originating within the nasal sinuses11.
Cultures of single-cell suspensions of NALT are not practical due to low yields of mononuclear cells. However, NALT biology can be examined by ex vivo culturing of the intact organ, and this method has the additional advantage of maintaining the natural tissue structure. For in vivo studies, genetic knockout models presenting defects limited to NALT are not currently available due to a poor understanding of the developmental pathway. For example, while lymphotoxin-&alpha; knockout mice have atrophied NALT, the Peyer's patches, peripheral lymph nodes, follicular dendritic cells and other lymphoid tissues are also altered in these genetically manipulated mice12,13. As an alternative to gene knockout mice, surgical ablation permanently eliminates NALT from the nasal passage without affecting other tissues. The resulting mouse model has been used to establish relationships between NALT and immune responses to vaccines1,3. Serial collection of serum, saliva, nasal washes and vaginal secretions is necessary for establishing the basis of host responses to vaccination, while immune responses originating directly from NALT can be confirmed by tissue culture. The following procedures outline the surgeries, tissue culture and sample collection necessary to examine local and systemic humoral immune responses to intranasal (IN) vaccination.

Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue NALT in Mouse Responses to Vaccines

Methods to examine contributions of the nasopharyngeal-associated lymphoreticular tissues (NALT) to nasal and systemic immune responses of mice to intranasal vaccines are described. We demonstrate a surgical procedure to establish a NALT-dependent mouse model and ex vivo cultures of extracted NALT.

鼻咽癌相关淋巴组织（NALT）在鼠标的响应中的作用研究疫苗

The nasopharyngeal-associated lymphoreticular tissues (NALT) found in humans, rodents, and other mammals, contribute to immunity in the nasal sinuses1-3. ...

科研

免疫与感染

A Quantitative Evaluation of Cell Migration by the Phagokinetic Track Motility Assay

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular organisms towards a source of nutrients or away from unsuitable conditions, as well as in multicellular organisms for tissue development, immune surveillance and wound healing, just to mention a few roles1,2,3. Deregulation of this process can lead to serious neurological, cardiovascular and immunological diseases, as well as exacerbated tumor formation and spread4,5. Molecularly, actin polymerization and receptor recycling have been shown to play important roles in creating cellular extensions (lamellipodia), that drive the forward movement of the cell6,7,8. However, many biological questions about cell migration remain unanswered.
The central role for cellular motility in human health and disease underlines the importance of understanding the specific mechanisms involved in this process and makes accurate methods for evaluating cell motility particularly important. Microscopes are usually used to visualize the movement of cells. However, cells move rather slowly, making the quantitative measurement of cell migration a resource-consuming process requiring expensive cameras and software to create quantitative time-lapsed movies of motile cells. Therefore, the ability to perform a quantitative measurement of cell migration that is cost-effective, non-laborious, and that utilizes common laboratory equipment is a great need for many researchers.
The phagokinetic track motility assay utilizes the ability of a moving cell to clear gold particles from its path to create a measurable track on a colloidal gold-coated glass coverslip9,10. With the use of freely available software, multiple tracks can be evaluated for each treatment to accomplish statistical requirements. The assay can be utilized to assess motility of many cell types, such as cancer cells11,12, fibroblasts9, neutrophils13, skeletal muscle cells14, keratinocytes15, trophoblasts16, endothelial cells17, and monocytes10,18-22. The protocol involves the creation of slides coated with gold nanoparticles (Au&deg;) that are generated by a reduction of chloroauric acid (Au3+) by sodium citrate. This method was developed by Turkevich et al. in 195123 and then improved in the 1970s by Frens et al.24,25. As a result of this chemical reduction step, gold particles (10-20 nm in diameter) precipitate from the reaction mixture and can be applied to glass coverslips, which are then ready for use in cellular migration analyses9,26,27.
In general, the phagokinetic track motility assay is a quick, quantitative and easy measure of cellular motility. In addition, it can be utilized as a simple high-throughput assay, for use with cell types that are not amenable to time-lapsed imaging, as well as other uses depending on the needs of the researcher. Together, the ability to quantitatively measure cellular motility of multiple cell types without the need for expensive microscopes and software, along with the use of common laboratory equipment and chemicals, make the phagokinetic track motility assay a solid choice for scientists with an interest in understanding cellular motility.

The phagokinetic motility track assay is a method used to assess the movement of cells. Specifically, the assay measures chemokinesis (random cell motility) over time in a quantitative manner. The assay takes advantage of the ability of cells to create a measurable track of their movement on colloidal gold-coated coverslips.

细胞迁移的的Phagokinetic轨道运动MLCK的定量评价

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular ...

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

定性和定量测定为检测和蛋白质抗菌药物的表征

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

A RAPID Method for Blood Processing to Increase the Yield of Plasma Peptide Levels in Human Blood

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the correct determination of a peptide&#39;s concentration, it should be stable during blood processing. However, this is not the case for several peptides which are quickly degraded by endogenous peptidases. Recently, we developed a blood processing method employing Reduced temperatures, Acidification, Protease inhibition, Isotopic exogenous controls and Dilution (RAPID) for the use in rats. Here, we have established this technique for the use in humans and investigated recovery, molecular form and circulating concentration of food intake regulatory hormones. The RAPID method significantly improved the recovery for 125I-labeled somatostatin-28 (+39%), glucagon-like peptide-1 (+35%), acyl ghrelin and glucagon (+32%), insulin and kisspeptin (+29%), nesfatin-1 (+28%), leptin (+21%) and peptide YY3-36 (+19%) compared to standard processing (EDTA blood on ice, p&#160;&#60;0.001). High performance liquid chromatography showed the elution of endogenous acyl ghrelin at the expected position after RAPID processing, while after standard processing 62% of acyl ghrelin were degraded resulting in an earlier peak likely representing desacyl ghrelin. After RAPID processing the acyl/desacyl ghrelin ratio in blood of normal weight subjects was 1:3 compared to 1:23 following standard processing (p&#160;= 0.03). Also endogenous kisspeptin levels were higher after RAPID compared to standard processing (+99%, p&#160;= 0.02). The RAPID blood processing method can be used in humans, yields higher peptide levels and allows for assessment of the correct molecular form.

The RAPID blood processing method can be used in humans and yields higher peptide levels as well as allows for assessment of the correct molecular form. Therefore, this method will be a valuable tool in peptide research.

对于血液处理快速的方法，以提高血浆肽水平的人血产量

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the ...

Assays for Studying the Role of Vitronectin in Bacterial Adhesion and Serum Resistance

Bacteria utilize complement regulators as a means of evading the host immune response. Here, we describe protocols for evaluating the role vitronectin acquisition at the bacterial cell surface plays in resistance to the host immune system. Flow cytometry experiments identified human plasma vitronectin as a ligand for the bacterial receptor outer membrane protein H of Haemophilus influenzae type f. An enzyme-linked immunosorbent assay was employed to characterize the protein-protein interactions between purified recombinant protein H and vitronectin, and binding affinity was assessed using bio-layer interferometry. The biological importance of the binding of vitronectin to protein H at the bacterial cell surface in evasion of the host immune response was confirmed using a serum resistance assay with normal and vitronectin-depleted human serum. The importance of vitronectin in bacterial adherence was analyzed using glass slides with and without vitronectin coating, followed by Gram staining. Finally, bacterial adhesion to human alveolar epithelial cell monolayers was investigated. The protocols described here can be easily adapted to the study of any bacterial species of interest.

This report describes protocols for characterizing interactions between bacterial outer membrane proteins and the human complement regulator vitronectin. The protocols can be used to study the binding reactions and biological function of vitronectin in any bacterial species.

Vitronectin 在细菌黏附和血清耐药性中作用的实验研究

Bacteria utilize complement regulators as a means of evading the host immune response. Here, we describe protocols for evaluating the role vitronectin ...

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention. This protocol focuses on three hallmark tissues studied in obesity and atherosclerosis including the liver, white adipose tissues (WAT), and the aorta. We highlight how dualistic usage of flow cytometry can achieve a new dimension of isolation and characterization of tissue-resident macrophages. A fundamental section of this protocol addresses the intricacies underlying tissue-specific enzymatic digestions and macrophage isolation, and subsequent cell-surface antibody staining for flow cytometric analysis. This protocol addresses existing complexities underlying fluorescent-activated cell sorting (FACS) and presents clarifications to these complexities so as to obtain broad range characterization from adequately sorted cell populations. Alternate enrichment methods are included for sorting cells, such as the dense liver, allowing for flexibility and time management when working with FACS. In brief, this protocol aids the researcher to evaluate macrophage heterogeneity from a multitude of inflamed tissues in a given study and provides insightful troubleshooting tips that have been successful for favorable cellular isolation and characterization of immune cells in DIO-mediated inflammation.

This protocol allows a researcher to isolate and characterize tissue-resident macrophages in various hallmark inflamed tissues extracted from diet-induced models of metabolic disorders.

受肥胖相关炎症组织的巨噬细胞的分离、鉴定和纯化

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. ...

Isolation and Characterization of Extracellular Vesicles Produced by Iron-limited Mycobacteria

Mycobacteria, including Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, naturally release extracellular vesicles (EVs) containing immunologically active molecules. Knowledge regarding the molecular mechanisms of vesicle biogenesis, the content of the vesicles, and their functions at the pathogen-host interface is very limited. Addressing these questions requires rigorous procedures for isolation, purification, and validation of EVs. Previously, vesicle production was found to be enhanced when M. tuberculosis was exposed to iron restriction, a condition encountered by Mtb in the host environment. Presented here is a complete and detailed protocol to isolate and purify EVs from iron-deficient mycobacteria. Quantitative and qualitative methods are applied to validate purified EVs.

Mycobacterium tuberculosis shows increased production and release of extracellular vesicles in response to low iron conditions. This work details a protocol for generating low iron conditions and methods for the purification and characterization of mycobacterial extracellular vesicles released in response to iron deficiency.

铁限分枝杆菌产生的细胞外囊泡的分离和特征

Mycobacteria, including Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, naturally release extracellular vesicles (EVs) ...

Visualization, Quantification, and Mapping of Immune Cell Populations in the Tumor Microenvironment

The immune landscape of the tumor microenvironment (TME) is a determining factor in cancer progression and response to therapy. Specifically, the density and the location of immune cells in the TME have important diagnostic and prognostic values. Multiomic profiling of the TME has exponentially increased our understanding of the numerous cellular and molecular networks regulating tumor initiation and progression. However, these techniques do not provide information about the spatial organization of cells or cell-cell interactions. Affordable, accessible, and easy to execute multiplexing techniques that allow spatial resolution of immune cells in tissue sections are needed to complement single cell-based high-throughput technologies. Here, we describe a strategy that integrates serial imaging, sequential labeling, and image alignment to generate virtual multiparameter slides of whole tissue sections. Virtual slides are subsequently analyzed in an automated fashion using user-defined protocols that enable identification, quantification, and mapping of cell populations of interest. The image analysis is done, in this case using the analysis modules Tissuealign, Author, and HISTOmap. We present an example where we applied this strategy successfully to one clinical specimen, maximizing the information that can be obtained from limited tissue samples and providing an unbiased view of the TME in the entire tissue section.

Here, we describe a simple and accessible strategy for visualizing, quantifying, and mapping immune cells in formalin-fixed paraffin-embedded tumor tissue sections. This methodology combines existing imaging and digital analysis techniques with the purpose of expanding the multiplexing capability and multiparameter analysis of imaging assays.

肿瘤微环境下免疫细胞种群的可视化、定量和映射

The immune landscape of the tumor microenvironment (TME) is a determining factor in cancer progression and response to therapy. Specifically, the density ...

Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant advantage over other methods. To prepare these probes, azide derivatives of antibiotics are synthesized, then coupled with alkyne-fluorophores using azide-alkyne dipolar cycloaddition by click chemistry. Following purification, the antibiotic activity of the fluorescent antibiotic is tested by minimum inhibitory concentration assessment. In order to study bacterial accumulation, either spectrophotometry or flow cytometry may be used, allowing for much simpler analysis than methods relying on radioactive antibiotic derivatives. Furthermore, confocal microscopy can be used to examine localization within the bacteria, affording valuable information about mode of action and changes that occur in resistant species. The use of fluorescent antibiotic probes in the study of antimicrobial resistance is a powerful method with much potential for future expansion.

Fluorescently tagged antibiotics are powerful tools that can be used to study multiple aspects of antimicrobial resistance. This article describes the preparation of fluorescently tagged antibiotics and their application to studying antibiotic resistance in bacteria. Probes can be used to study mechanisms of bacterial resistance (e.g., efflux) by spectrophotometry, flow cytometry, and microscopy.

使用荧光抗生素探针对细菌耐药性的可视化

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant ...

Characterization of Immune Cells and Proinflammatory Mediators in the Pulmonary Environment

Immune cell expansion, activation, and trafficking to the lungs, which are controlled by the expression of multiple cytokines and chemokines, may be altered by severe brain injury. This is evidenced by the fact that pneumonia is a major cause of mortality in patients who have suffered from ischemic stroke. The goal of this protocol is to describe the use of multicolor flow cytometric analysis to identify 13 types of immune cells in the lungs of mice, including alveolar macrophages, interstitial macrophages, CD103+ or CD11b+ dendritic cells (DCs), plasmacytoid DCs, eosinophils, monocytes/monocyte-derived cells, neutrophils, lymphoid-derived&#160; T and B cells, NK cells, and NKT cells, following ischemic stroke induction by transient middle cerebral artery occlusion. Moreover, we describe the preparation of lung homogenates using a bead homogenization method, to determine the expression levels of 13 different cytokines or chemokines simultaneously by multiplex bead arrays coupled with flow cytometric analysis. This protocol can also be used to investigate the pulmonary immune response in other disease settings, such as infectious lung disease or allergic disease.

This protocol describes the use of flow cytometry to identify the changes in immune cell composition, cytokine profile, and chemokine profile in the pulmonary environment following transient middle cerebral artery occlusion, a murine model of ischemic stroke.

肺环境中免疫细胞和促炎介质的表征

Immune cell expansion, activation, and trafficking to the lungs, which are controlled by the expression of multiple cytokines and chemokines, may be ...