我们要感谢他们的技术援助布兰登哈塞特和卡洛斯·奥尔蒂斯。这项工作是由国家科学基金会资助的IOB-0544428，内部监督办公室-0951272，的IOS-0925561博士迈克尔Kolomiets的支持，由美国农业部国家食品和农业研究所（粮农），AFRI植物育种和教育补助金＃2010-85117 -20539到博士。赛斯美利，托马斯Isakeit，和迈克尔Kolomiets的。

1。玉米内核生物活性的<ol><li>两个星期前，文化马铃薯葡萄糖琼脂（PDA）的病原真菌在28°C。 </li><li>选择具有类似的形状和大小的内核，最好是扁平的，所以他们打下水平的生物活性瓶的底部，并在50毫升猎鹰管。内核选择必须已同时在相同的环境，以确保类似种子的年龄和代谢产物的组成。 </li><li>晃动管在室温下用70％乙醇，用无菌水1分钟，10分钟，5分钟与6％次氯酸钠表面消毒内核。冲洗，三次，每次5分钟，用无菌水，同时继续晃动。八内核在蒸压毛巾干。 </li><li>为了方便黄曲霉感染，创建一个地下18针深度为0.5厘米的胚胎端上的小伤口。然而，在我们的经验，需要一个较大的伤口镰刀菌感染。日erefore，用刀片切割深度为0.5毫米到胚胎。 </li><li>准备刮约5毫升的蒸压0.01％Tween-20的解决方案，以解放孢子和由重力通过至少4层灭菌纱布过滤除去菌丝体培养板接种暂停。孢子浓度与血球计算和调整到10 6个孢子/毫升为A。黄曲霉或F。 verticillioides。如果使用其他种类的病原体或主机，它是重要的，以评估适当的感染接种浓度。 </li><li>建立在一个塑料容器内衬5张纸巾和100毫升无菌水（29.2厘米×18.7厘米×8.3厘米）的湿度室。商会是不是水或气密性的和额外的水应加在整个实验，以保持湿润的纸巾。 </li><li>地方蒸压20毫升玻璃闪烁瓶和四核记录它们的质量。接种200μL孢子悬浮液，帽，涡悬浮均匀大衣内核。拧开瓶盖，自由允许到湿度室的空气交换和地方。 </li></ol> 注：在这些方法中所描述的其他种子或真菌，接种所需金额可能有所不同，并应得到实验。 <ol start="8"><li>孵育下12-h-light/12-h-dark光照在29°C为7天，或直至所需的内核。 </li></ol> 2。分生孢子枚举<ol><li>感染期后，加入5毫升蒸压0.01％Tween-20的闪烁小瓶含1分钟彻底感染的内核和涡。 </li><li>使用大口径的枪头，立即转移到2.0毫升含0.01％Tween-20的（或稀释需要取决于对感染）1.8毫升的Eppendorf管两个独立的孢子悬浮液200μL等份稀释。 </li><li>枚举每200μL等分两次与血球4比较处理间的的分生孢子水平。 </li></ol> 3。黄曲霉毒素定量<ol><li>从1个样本，以50毫升果汁杯20毫升80％的甲醇和0.05克氯化钠，1分钟盖和高速混合添加内核。 </li><li> 1凹槽滤纸置于干净的收集容器，倒入过滤器提取。 </li><li> 10毫升的滤液转移到一个干净的容器，加入20毫升蒸馏水H 2 O，并拌匀。 </li><li>通过1.5微米的玻璃微纤维过滤到一个干净的杯子的过滤器样品。 </li><li>申请黄曲霉测试列和过滤提取液1毫升，用压缩空气，强行通过列1-2滴/秒，直到清空率的过滤提取。 </li><li> 1毫升的蒸馏水2两次洗列，并通过列在1-2滴/秒，直到空气来通过传递。 </li><li> 1毫升100％HPLC级甲醇在1-2滴/秒的速率将玻璃试管收集流出列。 </li><li>加1毫升的的黄曲霉测试开发到洗脱液，拌匀。地方试管校准荧光和读取60秒。 </li></ol> 注：当使用的Aflatest边检总站协议，从荧光的测量计算后，根据最初的50克在100毫升中提取样品。如果考虑1毫升稀释的水样，一列代表0.166克的样品。因此，修改协议时，你需要考虑到的样本大小的差异和初始提取液。例如，内核2克20毫升80％的甲醇中提取。这是0.1克/毫升。当此示例1毫升，2毫升的水混合，样品在液体中的比例是0.033克/毫升。如果这个样品给出了100 ppb的荧光读数，实际样本的比例为0​​.166，也就是说，PPB =（0.166革兰氏×100 PPB）/ 0.1革兰氏），或166 ppb的浓度是基于。替代黄曲霉quantifi的阳离子的方法，请参考7。 4。伏马菌素B1（FB1的）分析<ol><li>当玉米种植木耳，抽取样品10毫升的乙腈/水（50/50，V / V），在室温无搅拌过夜。随后，与去离子水（6毫升）混合提取物（2毫升），适用本混合（8毫升）直接的C-18固相萃取。 </li><li>加载样品之前，先决条件的C-18固相萃取柱2毫升乙腈2毫升的水冲洗。 </li><li>装入样品后，列洗净，用2毫升水2毫升的乙腈/水（15/85，V / V）。 2毫升的乙腈/水（70/30，V / V）洗脱高效液相色谱分析样品（含FB1的）。 </li><li> Derivati​​ze与邻phthaldehyde的甲醛（OPA）0.1毫升柱洗脱液转移到小瓶含0.1毫升硼酸盐缓冲液（0.05 mol硼酸acid/0.05中号硼酸钠[50/50，V / V]，pH值8.5）和0.1 FB1的OPA的毫升（0.1 mg / ml的丙酮itrile与0.5％ - 巯基乙醇）。 </li><li>中号acetonitrile/0.01的硼酸（40/60，V / V）加入0.5毫升，10分钟后停止反应。 </li><li>分析FB1的岛津高效液相色谱LC-20AT配备的分析ZORBAX ODS柱（4.6 150毫米）和可变波长Shimatzu RF-10Axl荧光检测器（激发波长335纳米/ 440 nm处发射）系统。 </li><li>使用线性渐变（溶剂A：乙腈中号磷酸钠（40/60），pH值3.3;溶剂B：乙腈/ 0.1 M磷酸钠（60/40），pH值3.3）和梯度程序如下： 100％到100％B级10分钟，5分钟为100％乙。 </li><li> FB1的标准高效液相色谱法分析，峰面积测量用于生成标准曲线。随后，量化与FB1的标准曲线的峰面积FB1样品中的水平。 </li></ol> 5。麦角固醇分析<ol><li>文化的真菌玉米为7-14天。 </li><li>潜伏期后，加入10毫升氯仿：冰毒anol（2:1，V / V），在每个小瓶。一旦加入，以及动摇样品，然后在室温下孵化24小时在黑暗中的小瓶。 </li><li> 24小时后，离心样品，收集上清，通过0.45微米尼龙膜过滤。 </li><li>样品直接注入到岛津高效液相色谱LC-20AT，配备了4.6üODS-C18柱（200，250±4.6毫米），岛津SPD-20A紫外/可见光检测器设置在282 nm处监测系统。 </li><li>使用甲醇（100％），在1.5毫升/分钟为流动相流速。通过比较样品峰面积从HPLC级麦角固醇生成标准曲线确定样品中麦角甾醇的定量。 </li></ol> 6。代表结果继接种和潜伏期为两到三天的湿度试验箱，霉菌的生长开始出现的内核。七天治疗后，治疗植物的营养生长，应该是清晰可见，whilé模拟控制应该是未受感染者（ 图1B，顶部）。潜伏期较长的时期，有利于更丰富的营养生长（ 图1B，底部）。此处所述的条件下（ 图2G），A. 黄曲霉野生型NRRL 3357显示8，6，8天，分别定植，黄曲霉毒素的积累，和分生孢子生产的最高值（ 图2，AC）。然而，当霉菌毒素污染和产孢单位的麦角甾醇相比，最大的水平，观察在4和第6天，分别为（ 图2）。图2F总结这些真菌生物量的依赖性最大价值的意见。有趣的是，在接种后4-6天之间，内核经过，看到的时间过程（ 图2H，底部）从样品中的总RNA的核酸降解。 几项研究，包括我们自己，有examin编号verticilliodies感染2,8,9,10,11,12玉米粒，并成功地用于观测时间点为7-13天。使用方法外，烟曲霉毒素水平范围从3,500-8,000纳克/克内核4,13和麦角甾醇含量范围从5,000-10,000纳克/克内核14,13。伏马菌素（上）和麦角甾醇（下图3显示了代表峰）高效液相色谱。麦角甾醇的测定，也可以进行通过吸收光谱15。 <img alt="图1" src="/files/ftp_upload/3727/3727fig1.jpg" /> 图1。真菌生物量，产孢霉菌毒素生产和代表从内核生物测定的营养生长结果的量化流程图。一）流程图概述了用来评估对玉米籽粒的真菌发病的基本生物学参数的定量描述方法。 b）代表为核心的生物测定的结果。顶，A。黄曲霉 （NRRL3357）在B73的遗传背景，对玉米籽粒的营养生长。种子接种200μL10 6个孢子/ mL和图片被接种后7天内。底部， 楼verticillioides接种B73的背景下13天感染后的内核。内核接种200μL10 6孢子/毫升。 <img alt="图2" src="/files/ftp_upload/3727/3727fig2.jpg" /> 图2。黄曲霉内核生物测定的时间过程。 B73的内核与200 10 6个孢子/毫升黄曲霉菌孢子悬浮液接种和培养2-8天（G）的。所有的值，确定从4粒干重平均（N = 3-4;均值±标准差）。一）定植（麦角甾醇），（二）黄曲霉毒素，和（C）产孢使用上述方法进行量化。五）黄曲霉毒素和（F）的产孢显示作为一个通过H麦角甾醇的测定真菌生物量的功能。 F）的观察麦角甾醇，黄曲霉毒素的积累，作为一种真菌生物功能的产孢的最高值 - 每个数量的最高值被设置为100％。 （八）1微克每车道从时间进程的总RNA。 <img alt="图3" src="/files/ftp_upload/3727/3727fig3.jpg" /> 图3。代表高高效液相色谱法（HPLC）为伏马菌素B1（顶部）和麦角甾醇（底部）从感染镰刀菌 （M3125）的内核分离的色谱图。

<ol>
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<p class="jove_content"&gt;我什么都没有透露。</p

<p class="jove_content"这里描述的方法已被广泛的测试和产生真菌孢子形成和霉菌毒素生产的量化结果被证明是强大的。此外，这些方法应该适用于从其他mycotoxigenic真菌（如花生，小麦，棉花，开心果等），很容易受到污染的植物物种的种子。主管植物病原菌互作分析，这是必须活着保存的种子。内核的胚胎方面的一个小伤口有利于感染，同时保持种子的活力。</p&gt;<p class="jove_content"有效运行的内核检测已被证明是来?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 产品编号 </td></tr><tr><td>马铃薯葡萄糖琼脂</td><td>费舍尔Scientifc </td><td> S71659A </td></tr><tr><td>吐温20 </td><td>费舍尔Scientifc </td><td> BP337-100 </td></tr><tr><td>塑料孵化容器</td><td> sterilite </td><td> 1713LAB06 </td></tr><tr><td>混合器</td><td> VICAM </td><td> 20200 </td></tr><tr><td> 24厘米凹槽滤纸</td><td> VICAM </td><td> 31240 </td></tr><tr><td> 1.5微米的玻璃微纤维</td><td> VICAM </td><td> 31955 </td></tr><tr><td>黄曲霉试验柱</td><td> VICAM </td><td> G1024 </td></tr><tr><td> afrla测试开发人员</td><td> VICAM </td><td> 32010 </td></tr><tr><td>甲醇</td><td> VICAM </td><td> 35016 </td></tr><tr><td>乙腈</td><td>费舍尔Scientifc </td><td> AC14952-0025 </td></tr><tr><td>乙醇</td><td>费舍尔王欧，吴文良TIFC </td><td> AC39769-0025 </td></tr><tr><td>的C-18固相萃取柱（PREP SEP SPE C18柱） </td><td>费舍尔Scientifc </td><td> 60108-304 </td></tr><tr><td>邻苯二甲醛（OPA）的</td><td>西格玛化工有限公司</td><td> 79760-5G </td></tr><tr><td>硼酸</td><td>费舍尔Scientifc </td><td> BP168-500 </td></tr><tr><td>四硼酸钠</td><td>费舍尔Scientifc </td><td> RDCS0330500 </td></tr><tr><td>巯基乙醇</td><td>费舍尔Scientifc </td><td> 45-000-231 </td></tr><tr><td>岛津高效液相色谱LC-20AT（泵） </td><td>岛津科学仪器公司</td><td> LC-20AT </td></tr><tr><td> ZORBAX ODS柱（4.6x150mm） </td><td>安捷伦科技公司</td><td> 443905-902 </td></tr><tr><td> shimatzu RF-10Axl荧光检测</td><td>岛津科学仪器公司</td><td> RF-10AXL </td></tr><tr><td>磷酸钠</td><td>费舍尔Scientifc </td><td> AC38987-0010 </td></tr><tr><td> FB1的标准</td><td> Sigma公司</td><td> F1147-1毫克</td></tr><tr><td>氯仿</td><td>厂商VWR </td><td> MK444410 </td></tr><tr><td>注射器过滤器13毫米与0.45微米的尼龙膜法（HPLC） </td><td>颇尔生命科学</td><td> 4426 </td></tr><tr><td>麦角甾醇</td><td> Sigma-Aldrich公司</td><td> 45480-50G - F </td></tr><tr><td>闪烁瓶</td><td>厂商VWR </td><td> 66021-602 </td></tr><tr><td>氯化钠</td><td> VICAM </td><td> G1124 </td></tr></tbody></table>

quantification of fungal colonization, sporogenesis, and production of mycotoxins using kernel bioassays

由种子腐烂粮食感染真菌对全球谷物产量最大的经济挑战之一，更不用说对人类和动物健康的严重风险。其中谷物产量中，玉米无疑是受影响最严重的作物，由于粮食的完整性和霉菌毒素的种子污染的病原体引起的损失。玉米种植者和食品和饲料加工两个最普遍的问题霉菌毒素是黄曲霉毒素和伏马毒素， 黄曲霉菌和镰刀菌产生，分别。 分子植物-病原体相互作用的最近的研究已经证明，在了解相关的具体机制与植物的真菌感染和真菌毒素污染1,2,3,4,5,6的承诺。因为许多实验室正在使用的内核分析，研究植物病原体相互作用，有需要为不同的生物参数量化标准的方法，所以来自不同实验室的结果可以交叉进行解释。一个强大的和可重复性的种子的定量分析手段，我们已经制定了在实验室内核检测和随后的方法，以量化霉菌的生长，生物量和霉菌毒素污染。四消毒玉米籽粒真菌悬液（10 6）接种在玻璃小瓶和培养一个预定的期限内。样品瓶，然后选择由血球，麦角甾醇的生物分析，高效液相色谱（HPLC），黄曲霉毒素定量使用AflaTest荧光法，高效液相色谱法和伏马菌素量化为分生孢子枚举。

Watch this Scientific Journal Video about Quantification of Fungal Colonization, Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays at JoVE.com

Quantification of Fungal Colonization, Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays

谷类作物种子感染真菌的破坏，促使大量的研究工作，以便更好地了解植物病原体相互作用。研究种子真菌的相互作用，在实验室环境中，我们开发了一个强大的真菌繁殖，生物质能和使用内核生物测定霉菌毒素污染的量化方法。

使用内核生物测定真菌孢子发生和霉菌毒素生产的定量

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks ...

quantification-fungal-colonization-sporogenesis-production-mycotoxins

Research

JoVE Journal

Immunology and Infection

18.0K 次观看.  Texas A&M University. 谷类作物种子感染真菌的破坏，促使大量的研究工作，以便更好地了解植物病原体相互作用。研究种子真菌的相互作用，在实验室环境中，我们开发了一个强大的真菌繁殖，生物质能和使用内核生物测定霉菌毒素污染的量化方法。

视频: Quantification of Fungal Colonization, Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays

Colonization of Euprymna scolopes Squid by Vibrio fischeri

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

Colonization of Euprymna scolopes Squid by Vibrio fischeri

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

定植 Euprymna scolopes鱿鱼由弧菌鲵</em

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

科研

免疫与感染

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 ...

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates2, or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae infection models.
Our mouse model of intranasal colonization is adapted from human models3 and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx4-7. In the first part of the model, we use a clinical isolate of S. pneumoniae to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice8, however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.

肺炎链球菌在鼻内殖民期间炎症反应的特征

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing ...

Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or pathogenic effects depending on the inflammatory milieu. Nonetheless, despite the indispensable role of neutrophils in immunity, detailed understanding of the molecular factors that mediate neutrophils' effector and immunopathogenic effects in different infectious diseases and inflammatory conditions is still lacking, partly because of their short half life, the difficulties with handling of these cells and the lack of reliable experimental protocols for obtaining sufficient numbers of neutrophils for downstream functional studies and adoptive transfer experiments. Therefore, simple, fast, economical and reliable methods are highly desirable for harvesting sufficient numbers of mouse neutrophils for assessing functions such as phagocytosis, killing, cytokine production, degranulation and trafficking. To that end, we present a reproducible density gradient centrifugation-based protocol, which can be adapted in any laboratory to isolate large numbers of neutrophils from the bone marrow of mice with high purity and viability. Moreover, we present a simple protocol that uses CellTracker dyes to label the isolated neutrophils, which can then be adoptively transferred into recipient mice and tracked in several tissues for at least 4 hr post-transfer using flow cytometry. Using this approach, differential labeling of neutrophils from wild-type and gene-deficient mice with different CellTracker dyes can be successfully employed to perform competitive repopulation studies for evaluating the direct role of specific genes in trafficking of neutrophils from the blood into target tissues in vivo.

We describe a protocol for isolation and purification of neutrophils from mouse bone marrow by density gradient centrifugation and for neutrophil labeling using CellTracker dyes. This represents a simple, fast, reproducible and economical method for obtaining large numbers of neutrophils for downstream functional studies or adoptive transfer and tracking experiments.

功能研究和过继转移实验小鼠骨髓嗜中性粒细胞的分离，纯化及贴标

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

建立一个<em&gt;在体外</em&gt;系统来研究细胞内的行为<em&gt;光滑念珠菌</em&gt;在人THP-1巨噬细胞

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal&#160;colonization.&#160;The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

使用一个调查益生素对肺炎球菌定植的影响<em&gt;体外</em&gt;粘附试验

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised patients, the elderly, and pregnant women. The most common manifestations of listeriosis in humans include meningitis, encephalitis, and fetal abortion. A significant but much less documented sequelae of invasive L. monocytogenes infection involves the heart. The death rate from cardiac illness can be up to 35% despite treatment, however very little is known regarding L. monocytogenes colonization of cardiac tissue and its resultant pathologies. In addition, it has recently become apparent that subpopulations of L. monocytogenes have an enhanced capacity to invade and grow within cardiac tissue. This protocol describes in detail in vitro and in vivo methods that can be used for assessing cardiotropism of L. monocytogenes isolates. Methods are presented for the infection of H9c2 rat cardiac myoblasts in tissue culture as well as for the determination of bacterial colonization of the hearts of infected mice. These methods are useful not only for identifying strains with the potential to colonize cardiac tissue in infected animals, but may also facilitate the identification of bacterial gene products that serve to enhance cardiac cell invasion and/or drive changes in heart pathology. These methods also provide for the direct comparison of cardiotropism between multiple L. monocytogenes strains.

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes causes fetal infections in pregnant women and meningitis in susceptible populations. Subpopulations of bacteria can colonize cardiac tissue, causing myocarditis in patients and laboratory animals. Here we present a protocol that describes how to assess L. monocytogenes cardiac cell invasion in vitro and cardiac colonization in infected animals.

使用李斯特菌单细胞基因评估受感染小鼠培养和心脏殖民化中心脏细胞的细菌入侵

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

B组的小鼠模型<em&gt;链球菌</em&gt;阴道定植

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

Histological Quantification to Determine Lung Fungal Burden in Experimental Aspergillosis

The quantification of lung fungal burden is critical for the determination of the relative levels of immune protection and fungal virulence in mouse models of pulmonary fungal infection. Although multiple methods are used to assess fungal burden, quantitative polymerase chain reaction (qPCR) of fungal DNA has emerged as a technique with several advantages over previous culture-based methods. Currently, a comprehensive assessment of lung pathology, leukocyte recruitment, fungal burden, and gene expression in mice with invasive aspergillosis (IA) necessitates the use of a significant number of experimental and control animals. Here the quantification of lung histological staining to determine fungal burden using a reduced number of animals was examined in detail. Lung sections were stained to identify fungal structures with Gomori&#39;s modified methanamine silver (GMS) staining. Images were taken from the GMS-stained sections from 4 discrete fields of each formalin-fixed paraffin-embedded lung. The GMS stained areas within each image were quantified using an image analysis program, and from this quantification, the mean percentage of stained area was determined for each sample. Using this strategy, eosinophil-deficient mice exhibited decreased fungal burden and disease with caspofungin therapy, while wild-type mice with IA did not improve with caspofungin. Similarly, fungal burden in mice lacking &#947;&#948; T cells were also improved by caspofungin, as measured by qPCR and GMS quantification. GMS quantification is therefore introduced as a method for the determination of relative lung fungal burden that may ultimately reduce the quantity of experimental animals required for comprehensive studies of invasive aspergillosis.

Here we describe a protocol to determine pulmonary fungal burden in mice with invasive aspergillosis by quantification of Gomori&#39;s modified methanamine silver staining in histological sections. Use of this method resulted in comparable results with less animals compared to assessment of fungal burden by quantitative PCR of lung fungal DNA.

实验性曲霉病肺真菌负担的组织学定量测定

The quantification of lung fungal burden is critical for the determination of the relative levels of immune protection and fungal virulence in mouse ...

Real-time Imaging and Quantification of Fungal Biofilm Development Using a Two-Phase Recirculating Flow System

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. While models for the growth under flow have been developed, many of these systems are expensive, or do not allow imaging while the cells are under flow. We have developed a novel apparatus that allows us to image the growth and development of Candida albicans cells under flow and in real-time. Here, we detail the protocol for the assembly and use of this flow apparatus, as well as the quantification of data that are generated. We are able to quantify the rates that the cells attach to and detach from the slide, as well as to determine a measure of the biomass on the slide over time. This system is both economical and versatile, working with many types of light microscopes, including inexpensive benchtop microscopes, and is capable of extended imaging times compared to other flow systems. Overall, this is a low-throughput system that can provide highly detailed real-time information on the biofilm growth of fungal species under flow.

We describe the assembly, operation, and cleaning of a flow apparatus designed to image fungal biofilm formation in real time while under flow. We also provide and discuss quantitative algorithms to be used on the acquired images.

用两相循环流系统实时成像和定量真菌生物膜的研究

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. ...