我们感谢分子生物科学 (手机) 博士计划和中田纳西州立大学 (MTSU) 的生物学系为这项研究提供资金。该项目还部分由 MTSU 基金会授予 D.E.N. 的特别项目赠款提供经费。

注意: C. 隐球菌是生物安全级别 2 (BSL-2) 病原体, 研究人员必须采取适当的预防措施。有关如何与 BSL-2 病原体安全工作的详细程序, 可在疾病控制中心网站上找到, 但重要的是要注意, 所有接触到C. 隐球菌的人都应该接受适当的训练来处理致病剂应始终佩戴适当的个人防护用品 (PPE), 一般是乳胶或丁腈橡胶手套。此外, 离心机上的转子应密封, 以防止 aerosolization 的样品和任何泄漏清除立即使用10% 漂白剂解决方案28。
1. 胶囊诱导
注: 除非另行注明, 所有步骤均应在室温下进行。
<ol><li>在酵母蛋白胨葡萄糖 (YPD) 汤 (pH 值 6.5 +/-0.2) 中培养C. 隐球菌, 并在37摄氏度孵育, 直到它们处于对数阶段 (约 18-36 h)。</li>
	<li>离心细胞在 870 x g为5分钟在21°c 和洗涤三次与磷酸盐缓冲盐水 (PBS)。</li>
	<li>使用 hemocytometer (或自动单元格计数器) 在 Dulbecco 最小的基本介质 (DMEM) 中, 在 2 x 105 cells/2 mL 上计数单元格和并用重悬。</li>
	<li>诱导胶囊, 孵育在37°c 和 5% CO2为 18 h。 注意: 在介质中缺少营养素和 CO2的存在相结合会刺激C. 隐球菌胶囊的生长。</li>
	<li>孵化后, 通过离心 (如上所述) 收割细胞, 除去除5-10 µL 上清的全部。同时, 收获相应的非诱导细胞样本, 为每个菌株进行测量。使用这些作为负控制。 注: 细胞小球将非常小, 可能很难看到。吸上清液时要小心。</li>
	<li>用印度墨水 (2.1 节) 或荧光染料染色 (2.2 节)。</li>
</ol>2. 染色
<ol><li>
只用印度墨水染色
	<ol>
<li>为了用印度墨水染色酵母, 并用重悬在剩余的清液中的颗粒, 并将 4μL (大约一半) 的细胞悬浮物和4μL 的印度墨水添加到玻璃显微镜滑梯上。轻轻搅拌, 避免起泡。</li>
		<li>应用13毫米玻璃盖玻片 (#1 5 厚度) 和密封边缘与无毒密封胶 (清除指甲油), 以防止样品干燥。</li>
	</ol>
</li>
	<li>
只用荧光染料染色
	<ol>
<li>用荧光染料染色酵母, 并用重悬50µL PBS 中的细胞颗粒, 1% 牛血清白蛋白 (BSA)。</li>
		<li>用等量的 Calcofluor 白色 (CFW) (1 µg/毫升) 和蒴果单克隆 18B7 (参见材料表), 在37摄氏度的30分钟内, 孵化出与 AlexaFluor488 (AF488) (10 µg/毫升) 相结合的培养物。</li>
		<li>离心细胞在 870 x g 5 分钟在21°c 后孵化和吸出上清。</li>
		<li>并用重悬细胞颗粒在10µL PBS 与1% 牛血清白蛋白 (BSA)。</li>
		<li>吸管8µL 细胞悬浮在玻璃显微镜幻灯片上。</li>
		<li>应用13毫米玻璃盖玻片 (#1 5 厚度) 和密封边缘与无毒密封胶 (清除指甲油), 以防止样品干燥。</li>
	</ol>
</li>
	<li>
用印度墨水和荧光染料染色
	<ol>
<li>为了更直接地比较印度墨水染色与荧光染料的功效, 用两种着色剂对同一细胞种群进行染色。要做到这一点, 首先孵化细胞与荧光胶囊和细胞壁染色, 每步 2.2.1-2.2.3。</li>
		<li>接下来, 将荧光染色的电池悬浮液和印度墨水的等量容积 (4 µL) 移到玻璃滑梯上。轻轻地混合。</li>
		<li>应用13毫米玻璃盖玻片 (#1 5 厚度) 和密封的边缘与无毒密封胶 (清除指甲油), 以防止样品干燥。</li>
	</ol>
</li>
</ol>3. 图像采集/显微术
<ol><li>
用印度墨水染成的成像细胞。
	<ol>
<li>使用标准光显微镜 (100X 放大倍数) 获取图像。</li>
		<li>每个条件下至少有50个单元格的图像。 注意: 每个字段的单元格数会有所不同, 这是可以接受的。然而, 重要的是, 重叠的单元格保持在最低限度, 因为它们很难测量 (手动和自动软件)。对于这些实验, 图像获得了在深度16位, 曝光时间优化, 以最大限度地提高图像对比度, 同时尽量减少的模糊, 因为小的移动的细胞。</li>
	</ol>
</li>
	<li>
荧光染料染色的成像细胞
	<ol>
<li>通过荧光显微镜对细胞进行成像, 打开显微镜成像软件, 选择合适的显影激发和发射波长 (CFW 和 AF488 分别使用405nm 和 488 nm 灯激发)。使用荧光显微镜获取图像。</li>
		<li>每个条件下至少有50个单元格的图像。 注意: 每个字段的单元格数会有所不同, 这是可以接受的。然而, 重要的是, 重叠的单元格保持在最低限度, 因为这些都难以测量 (手动和自动软件)。</li>
		<li>获取单元格的 z 堆栈图像序列, 以确保获得给定字段中每个单元格的最大胶囊直径。<ol>
<li>在显微镜控制软件中, 点击 "z 栈" 栏打开 "z 栈" 控制面板, 然后选择面板左上角的 "第一个/最后一个" 选项。</li>
			<li>使用显微镜上的精细对焦控制, 集中在一个单一酵母细胞的最宽点以下的水平, 并在当前的视野中的所有胶囊, 并点击 "设置第一"。</li>
			<li>使用精细焦点控件将焦点放在同一个单元格体的最宽点之上, 并在当前字段中的所有单元格中, 单击 "最后设置"。</li>
			<li>然后, 单击 "获取" 选项卡上的 "开始实验" 以获取当前位置的 z 堆栈。在多个位置重复该过程, 直到获得至少50个单元格的 z 堆栈图像为止。 注: 对于这些实验, 共焦针孔被设置为 1.0 AU 和一个重叠 z 系列10-20 片覆盖0.7 µM 每一个在选定的位置获得。非通风单元。</li>
		</ol>
</li>
		<li>接下来, 使用适当的图像分析软件将每个 z 栈系列转换为最大强度投影 (MIP)。 注意: MIPs 项目在堆叠图像29的每一个平面上的最大强度。创建 MIPs 使一个人能够产生2维的图像表示形式, 对应于捕获的3维堆栈中的最大单元格和胶囊直径。</li>
	</ol>
</li>
	<li>
用印度墨水和荧光染料染成的成像细胞
	<ol>
<li>使用 widefield (40X 放大) 或共聚焦显微镜 (63X 或100X 放大) 获取图像。对于印有印度墨水和荧光染料的细胞, 使用 widefield 显微镜捕捉图像, 并配备计算机控制阶段和油浸泡目标。 注: 使用的显微镜应使用成像软件 CFW 控制。通过 340-380 nm 激发过滤器激发出的荧光, 发射的光应该通过从 400 nm 的分色镜中反射出来的 435-485 nm 屏障过滤器检测出来。AF488 荧光可以通过465-495 纳米励磁过滤器激发, 通过一个 505 nm 的分色镜反射的 515-555 nm 屏障过滤器可以检测出发射光。</li>
		<li>每个染色方法的每种条件下至少有50个细胞。</li>
	</ol>
</li>
</ol>4. 胶囊的手动测量
<ol><li>对于染有印度墨水或荧光污渍的细胞, 使用细胞测量软件手动测量胶囊和细胞直径 (参见材料表)。要做到这一点, 请转到 "文件" > "打开图像" > "度量值", 并使用游标在整个单元格的最宽点 (总直径) 中绘制一条直线。</li>
	<li>接下来, 再次单击 "测量", 并通过单元格体 (细胞体直径) 绘制一条直线。 注意: 测量被自动保存到单元格中。</li>
	<li>对所有单元格重复此过程 (最小为 50/组)。</li>
	<li>要在测量后保存图像, 请单击 "文件" > "另存为", 并相应地命名图像。</li>
	<li>选择新测量的文件并将数据导出到电子表格软件。</li>
	<li>用方程计算平均胶囊直径: ([总直径]-[细胞体直径])。</li>
</ol>5. 胶囊的自动测量
注意: 对于成功的自动测量, 请使用具有较高对比度的16位图像, 并将其与适当的图像分析软件适当地集中在一起 (请参见材料表)。当成像C. 隐球菌用于胶囊测量时, 重点放在细胞壁和胶囊之间的边界上的暗环上是很重要的。这使得软件能够正确地描绘细胞和胶囊。
<ol><li>使用适当的图像编辑软件反转并创建所有印有墨迹的彩色细胞图像的副本 (请参阅材料表)。为此, 请单击 "文件" > "打开" 以打开印度墨迹着色图像, 然后 "控制" + "I" 反转图像。单击 "文件" > "另存为" 以保存反转图像。</li>
	<li>要将原始图像和倒图导入到软件中, 请单击 "文件" > "导入序列" > "加载图像" > "定义序列 (手动)" > "维度 (通道)" > "导入" > "应用"。 注意: C. 隐球菌细胞体和胶囊可以用特定的算法 (称为 "食谱") 来识别和屏蔽, 该软件包括在内。</li>
	<li>使用 "菌落分析器 (荧光)" 食谱对倒置图像进行检测和掩模, 然后单击 "应用"。在反转图像中, 定义C. 隐球菌细胞边界的暗环比周围的胶囊更亮。</li>
	<li>使用 "菌落分析器 (荧光)" 食谱对倒置图像, 以分割C. 隐球菌细胞体从他们的胶囊, 然后单击 "应用"。这将创建计数掩码。通过右键单击标记为 "计数掩码" 的标签, 重命名此计数掩码 "单元格体"。 注意: 该食谱由多个图像处理步骤组成: 背景删除、对象检测、对象分离和子集筛选。</li>
	<li>接下来, 使用原始图像上的 "细胞增殖" 食谱来检测和遮盖胶囊, 然后点击 "应用"。这将创建计数掩码。通过右键单击标记为 "计数掩码" 的标签, 重命名此计数掩码 "胶囊"。</li>
	<li>使用食谱, ' 胶囊分区 ', 创建一个新的面具上的原始图像, 以分割相邻的胶囊彼此。为此, 请从食谱下拉菜单中单击 "胶囊分区"。在 "胶囊分区" 框中, 为胶囊区域选择现在重命名的计数掩码 "胶囊" (5.5)。为加密单元格选择现在重命名的计数掩码 "单元格体" (5.4)。为输入通道选择 "原件", 并 "为已分区的胶囊创建掩码。单击 "应用 注: 配方产生细胞和胶囊直径, 定义为穿过细胞和胶囊中心的最长和最短轴的平均值, 以及从检测掩模处测量的面积。查找此软件中 "电子表格" 选项卡下的输出。对所有图像重复此过程 (最少50个单元格/组)。该配方的参数可以调整, 以优化单个图像的检测或优化的批量检测多图像。额外的测量可以计算的配方, 综合表征细胞和胶囊的形态。</li>
	<li>将数据导出到电子表格软件的选择中进行进一步分析。</li>
</ol>

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P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+HAART+is+associated+with+decreased+risk+of+death+during+initial+treatment+of+cryptococcal+meningitis+in+adults+in+Botswana.">The use of HAART is associated with decreased risk of death during initial treatment of cryptococcal meningitis in adults in Botswana.</a> J Acquir Immune Defic Syndr. 49 (2), 227-229 (2008).</li><li>van Teeffelen, S., Shaevitz, J. W., Gitai, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image+analysis+in+fluorescence+microscopy:+bacterial+dynamics+as+a+case+study.">Image analysis in fluorescence microscopy: bacterial dynamics as a case study.</a> Bioessays. 34 (5), 427-436 (2012).</li><li>Granger, D. L., Perfect, J. R., Durack, D. 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作者 Hoyin, 是 DRVision 的产品经理, 出版了自动化图像分析软件 Aivia。

数十年来, 该胶囊一直是研究的主要焦点, 对 mycologists 和临床医生感兴趣的C. 隐球菌和球菌由于其作用的主要致病因素的病原体。用显微显微镜测量不同生长条件下的胶囊大小差异, 可以提供有关病原体及其对各种刺激的反应的重要信息 (即, 不同的环境条件,潜在的药物治疗,等) 在这里, 我们概述了一种方法, 以诱导生长的胶囊在C. 隐球菌, 并比较了两种不同的胶囊染色协议。最后, 我们展示了如何使用图像分析软件自动测量从印度墨水染色细胞的图像的胶囊直径, 产生的结果, 可比手工测量方法。
在我们的实验中, 我们比较了两个临床菌株, B18 和 B52, 与 H99S, 标准实验室应变的已知胶囊大小18。两种染色方法均表明, 临床菌株, B52, 有三株最大胶囊直径的检测。然而, 荧光测量的结果能够辨别出两株小胶囊大小的差异, 而印度的墨量测量则不同。这两种染色方法的结果是一致的, 这表明两者都是有效的, 但使用荧光染色的C. 隐球菌的图像可能会增加对小胶囊的应变量的敏感性。利用荧光染色细胞的 z 堆栈图像产生的最大投影, 研究人员可以确保即使成像细胞处于不同的焦平面, 也能精确地测量胶囊直径。这些测量对于使用印度墨水和标准光显微镜成像的C. 隐球菌来说更困难。在任何给定的图像中, 相邻的细胞可能会坐在略微不同的焦平面上, 从而难以准确地解决所有细胞的细胞体-胶囊边界。这可能会混淆调查人员检测胶囊大小或菌株之间细微差异的能力。
每种方法都有其固有的优点。印度墨水是最常用的研究人员, 因为它的负担能力, 易用性和稳定性 (它不会随着时间的推移, 像一些荧光污渍可以)。因为印度墨水是一个简单的, 负面的污点, 它可以与标准光显微镜结合使用, 这比荧光显微镜更容易获得。但是, 如果不使用正确的比例 (印度墨水: 缓冲区), 污点可以创建一个 "羽毛状" 的背景, 当映像, 这可能会使自动化图像分析复杂化。虽然使用昂贵和耗时更长的时间, 荧光染料可以有利于它们的灵活性, 而且正如我们所显示的, 它们的灵敏度。它们特别有用的测量胶囊大小和细胞负担的实验涉及c. 隐球菌已吞噬的免疫细胞, 是必要的组织学检查c. 隐球菌胶囊在组织示例 (本文中未概述的技术)32。如果用荧光染料染色, 如 calcofluor 白色或荧光标记的抗 GXM 抗体, 是有问题的 (例如, 染色不足), 这通常可以通过优化染色协议来克服 (增加/减少浓度和/或孵化时间与染料)。
无论染色方法如何, 我们发现胶囊诱导和正确的图像捕获是协议中的关键步骤。本文概述的协议是在C. 隐球菌中胶囊诱导的几种标准方法之一, 已成功地在酵母实验室中使用了多年23。C. 隐球菌胶囊感应是 CO2依赖, 只发生在压力条件下23,33。因此, 重要的是, 它发生在 5% CO2孵化器使用适当的营养缺乏生长培养基。如果怀疑在标准潜伏期 (一般为18-24 小时) 后未诱导胶囊, 则应将细胞与不暴露于压力条件的 uninduced 控制培养基进行比较。比较这两个将决定是否诱导是有效的。一个成功的诱导是大多数细胞制造一个更大的胶囊, 与 uninduced 控制相比。因为并不是所有的细胞都会诱发, 所以要测量的细胞应该是可用细胞的异构表示。
一致的图像采集的重要性不能夸大, 特别是如果自动化测量是目标。焦点、对比度、背景荧光以及文件的位深度和图像压缩都是重要的考虑因素。在可能的情况下, 不应避免在单个焦点平面上的大簇细胞, 尽管有些发芽和接触的细胞是可以接受的, 并且是不可避免的 (这条规则应遵循手动和自动测量)。在使用荧光染色细胞共聚焦成像时, 该问题可以在一定程度上规避。使用 z 堆栈来生成最大强度投影, 可以从单个图像29中精确测量不同焦平面中的单元格。此外, 在准备样品后, 图像应迅速获得。这对于印度墨水染色的样品来说尤其重要, 因为当它们开始干燥时, 背景和前景元素 (细胞) 之间的对比可能会减少, 不良的背景特征也可以增强, 从而降低自动化分割精度。
虽然胶囊直径测量在C. 隐球菌社区是司空见惯的, 直到这一点, 他们通常是手动执行的, 这需要大量的时间和人力。然而, 人工测量是在现场接受的, 结果可以以很少的费用获得。图像分析软件的进步使我们有可能采取的第一步, 以自动化这些测量。C. 隐球菌细胞体和胶囊在2D 图像中作为一个圆圈出现的简单性质使得测量它们的尺寸相对简单, 用于自动图像分割软件包, 而不需要开发新颖的算法.相反, 通过链接现有算法, C. 隐球菌细胞和胶囊可以被检测、分割和测量。使用这种方法, 我们已经自动测量了C. 隐球菌胶囊的直径, 这些细胞染色了最常用的胶囊染料, 印度墨水, 并比较了这些与手工测量相同的数据集。该方法提供了重现性的好处, 消除了与人工测量相关的人为误差, 同时节省了研究者的时间。此外, 在自动化分析工作流中, 可以将额外的测量结果纳入到最小的工作中。我们认为, 自动图像分析是一个有希望的方法, 使研究人员能够测量大量的C. 隐球菌胶囊具有高度的准确性和效率。
总的来说, 我们已经演示了如何在C. 隐球菌中成功诱导胶囊生长, 用印度墨水或荧光染料对样品进行染色, 并用人工和自动测量方法在图像分析中测量胶囊直径。软件.我们的研究结果表明, 这两种染色方法都是可行的, 通常产生一致的结果 (尽管印度墨水测量有更多的变异性)。此外, 手动和自动测量方法都有能力辨别不同的胶囊尺寸。

每年影响一季度百万人, 每年造成18万以上死亡,新生隐球菌是致病性, 胞内酵母和球菌1,2,的致病剂3. 受影响最严重的是贫穷国家的艾滋病毒阳性患者, 他们没有现成的抗逆转录病毒疗法, 使他们敏锐地易患疾病4,5,6。疾病控制中心的数据表明, 在撒哈拉以南非洲地区, 比起在记录中的 1上的任何埃博拉病毒爆发, 每年都有更多的人死于肺结核。最常见的接触途径是吸入在环境中常见的干燥孢子7。进入肺部后, 有几种致病因素有助于在受感染的个体中获得C. 隐球菌的成功。多糖胶囊被认为是微生物的主要毒力因子, 因为 acapsular 菌株不是毒性的8。
隐球菌胶囊由三个主要成分组成: glucuronoxylomannan (GXM)、galactoxylomannan (GalXM) 和 mannoproteins (MPs)9。虽然 MPs 是胶囊中相对较小的细胞壁相关组件, 但它们是免疫的, 可以促进一种主要支持炎症的响应9,10。相比之下, GXM 和 GalXM 构成了胶囊的大部分 (> 90% 的重量), 并有免疫抑制效应11。除了免疫调节作用外, 胶囊的快速扩增在体内形成了一个机械屏障, 可通过宿主吞噬细胞 (即、中性粒细胞和巨细胞)12摄取。C. 隐球菌胶囊及其合成是复杂的, 但总的来说, 增加的胶囊直径与增加的毒力6,13,14相关。考虑到这一点, 对于C. 隐球菌研究人员来说, 能够快速准确地量化胶囊测量是很重要的。
C. 隐球菌细胞及其多糖胶囊都是动态结构, 随着时间的推移15显示变化。该胶囊可以在密度、大小和组件上发生变化, 以响应主机环境中的更改16,17,18。低铁或营养水平, 接触血清, 人体生理 pH 值和增加 CO2是已知的启动胶囊生长16,18,19,20。此外, 研究人员还显示结构变化导致感染期间免疫反应有显著差异, 在其主机2122上为C. 隐球菌提供了优势。这是众所周知的, 因为C. 隐球菌胶囊的体系结构已经以多种方式进行了分析。例如, 电子显微镜显示, 该胶囊具有一个具有内部电子密层的异构矩阵, 在外层, 更透水层的下面是23。光散射和光镊的使用使研究人员得以进一步阐明其大分子特性24。通过对静态和动态光散射测量结果的分析, 我们知道多糖胶囊具有复杂的分支结构23。光学镊子用于测试结构的刚度, 并评估其抗体反应性24。然而, 到目前为止, 对C. 隐球菌胶囊最常用的分析是对其大小的测量。
为了量化胶囊的大小, 研究者使用了一个简单的测量方法: 胶囊的线性直径。数字显微镜用于捕获多个C. 隐球菌细胞 (一般上百个) 的图像, 它们都是印度墨水或荧光染料染色的。测量每个细胞体和周围胶囊的大小。通过对整个细胞直径 (细胞体 + 胶囊) 的减去细胞体直径, 计算出该胶囊的平均直径。直到这一点, 这些测量已经手动完成。虽然一般准确, 这种方法有缺点的研究人员。大型数据集可能需要几天甚至几周的时间来进行手工分析。由于这些测量是手工完成的, 所以主观和人为的错误可能会影响结果。
自动计算图像分析已成为许多分子细胞生物学领域的研究人员不可缺少的工具, 能够更快更可靠地分析生物图像25,26,27。精确的图像分析技术对于从通常复杂而庞大的数据集挖掘定量信息是必要的。然而, 一些测量, 特别是对C. 隐球菌胶囊的测量, 一直难以实现自动化。准确地识别细胞壁与胶囊之间的界面, 在相衬显微镜下成像时通常以暗环的形式出现, 用一个简单的阈值来解决会很麻烦。此外, 在文化中的C. 隐球菌细胞往往聚集在一起, 准确测量细胞的精确分割是必要的。
该项目的目的是 (i) 说明在C. 隐球菌中胶囊诱导的标准协议之一, (ii) 比较和对比印度墨水和荧光染色, 因为它们涉及胶囊直径测量, (iii) 发展简单,用印度墨水染色的图像测量胶囊直径的计算方法使用图像分析软件, (iv) 评估手动测量胶囊直径和使用软件自动化的好处和局限性。我们发现, 这两种染色方法, 荧光标记的细胞壁和胶囊, 而更费时, 提供了最一致的结果之间的实验。然而, 这两种方法都使我们能够成功地区分出不同胶囊大小的实验室和临床C. 隐球菌菌株。此外, 我们能够自动测量胶囊直径从印度墨水染色图像, 并发现这是一个可行的替代手工测量胶囊。

<table border="0" cellpadding="0" cellspacing="0" width="983">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:451px;">Capsule Induction</td>
			<td style="width:204px;"></td>
			<td style="width:117px;"></td>
			<td style="width:211px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">C. neoformans cells</td>
			<td></td>
			<td></td>
			<td>The clinical lab strain, H99S, was a kind gift from Dr. John Perfect (Duke University).&#160; The clinical strains, B18 and B52, were kind gifts from Dr. Greg Bisson (University of Pennsylania).&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yeast Peptone Dextrose Broth (YPD)</td>
			<td>Fisher Scientific</td>
			<td>DF0428-17-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate Buffered Saline (PBS)</td>
			<td></td>
			<td></td>
			<td>This is made in the lab using standard recipe (137mM NaCl, 2.7 mM KCl, 10mM Na2HPO4O, 2 mM Kh2PO4O)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM/high-glucose with L-glutamine, without sodium pyruvate</td>
			<td>GE Life Sciences</td>
			<td>SH30022.01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6-well plates</td>
			<td>Falcon</td>
			<td>CL5335-5EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Shaking incubator</td>
			<td>Thermo Scientific&#160;</td>
			<td></td>
			<td>MaxQ6000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CO2 incubator</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td>Isotemp</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td>Legend XTR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Staining</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microcentrifuge</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td>Legend Micro 21R</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">India ink</td>
			<td>Fisher Scientific</td>
			<td>14-910-56</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calcofluor white</td>
			<td>Sigma-Aldrich</td>
			<td>18909-100ML-F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">18B7 mouse anti-GXM antibody conjugated to Alexafluor 488</td>
			<td></td>
			<td></td>
			<td>A kind gift from Dr. Arturo Casadevall (Johns Hopkins University)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS with 1% Bovine Serum Albumin (BSA)</td>
			<td></td>
			<td></td>
			<td>PBS is the same recipe listed above (line 4) with 1% BSA added and filter sterilized.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A9418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Superfrost microscope slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-143</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass coverslips</td>
			<td>Corning</td>
			<td>2855-18</td>
			<td>#1.5 thickness</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Clear nail polish or other non-toxic sealant</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Image Acquisition&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immersion oil</td>
			<td>Cargille&#160;</td>
			<td>16484</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Light microscope with immersion oil objective</td>
			<td>Zeiss</td>
			<td></td>
			<td>Zeiss Axio A1 with a Plan - NEOFLUAR 100x oil immersion NA 1.30 objective</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Light microscope camera</td>
			<td>Zeiss</td>
			<td></td>
			<td>Zeiss Axiocam ErCD camera</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Confocal microscope with oil immersion objective</td>
			<td>Zeiss</td>
			<td></td>
			<td>LSM 700 laser scanning confocal equipped with a Plan-Apochromat 63X NA 1.4 oil immersion DIC M27 objective.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Confocal microscope software</td>
			<td>Zen 2009</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Confocal microscope camera</td>
			<td>Nikon</td>
			<td></td>
			<td>Nikon Ti-Eclipse with a Intensilight epifluorescence illuminator (Nikon), CoolSNAP MYO microscope camera (Photometrics), Plan Apo 60x NA 1.40 oil immersion objective (Nikon) and 1.5x magnification changer.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Widefield imaging software</td>
			<td>Nikon Elements (Nikon)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Capsule Measurement</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Image editing software</td>
			<td>Photoshop (Adobe)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microscope software for manual measurement</td>
			<td>Axiovision (Carl Zeiss)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Image analysis software for automated meesurement</td>
			<td>Aivia (DRVision Technologies)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spreadsheet software</td>
			<td>Excel (Microsoft)</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

size matters: measurement of capsule diameter in cryptococcus neoformans

新生隐球菌多糖胶囊是致病性酵母的主要毒力因子和最常见的研究领域之一。囊粒大小在菌株间有很大差异, 在引入压力或低养分条件时, 具有快速生长的能力, 与菌株的毒力呈正相关。由于这些原因, 胶囊的大小对C. 隐球菌研究人员非常感兴趣。在表型测试中诱导了C. 隐球菌胶囊的生长, 以帮助了解不同处理方法对酵母的影响以及菌株之间的大小差异。这里我们描述了胶囊诱导的标准方法之一, 并比较了两种被接受的染色和测量胶囊直径的方法: (i) 印度墨水, 一个负的污点, 使用与常规光镜和 (ii) 共染色与荧光染料的细胞壁和胶囊后, 共聚焦显微镜。最后, 我们展示了如何通过计算图像分析来自动测量来自印度墨水染色样品的胶囊直径。

为了说明胶囊诱导、细胞染色、影像学和测量技术, 我们使用了三株C. 隐球菌:常见的, 特点很好的实验室菌株, H99S30, 和前两个临床分离菌株未知的胶囊直径, B18 和 B5231。
使用印度墨水的胶囊感应、染色和图像采集工作流显示在图 1A中。从所有三种菌株中取出的图像在图 1B中显示为预置和后置。研究人员将知道, 如果胶囊诱导是成功的, 通过比较的细胞, 已经经历了胶囊诱导到那些从相同的文化, 没有。明显的大小增加通常是明显的在大多数菌株后诱导, 虽然一些菌株将展出自然小胶囊, 即使在诱导 (见图像的 B18 在图 1B)。应该指出的是, 如果在感应之前没有删除剩余的富媒体, 或者 CO2不存在, 胶囊感应将会失败。在这些情况下, 只有适度 (如果有) 增加的胶囊大小将被观察到。所有三株的直径都是手动测量的, 结果显示在图 1C中。在三的两个实验中, H99S 和 B18 的大小没有差别。但是, 在最大应变、B52 和 H99S 和 B18 (p < 0.001 和p < 0.001 分别为标准最小二乘法、样本大小 = 50 个细胞/应变的情况下, 一致地观察到大小的显著差异。).
图 2A描述了使用两种荧光染料 (一个用于胶囊 (18B7 AF488) 和一个用于细胞壁 (Calcofluor 白色) 的胶囊诱导、染色和图像采集工作流。图像被捕获为 z 栈的所有三菌株后诱导, 确保最大的胶囊和细胞体直径被捕获的每个细胞的测量 (图 2B)。每个 z 堆栈都经过处理以产生最大强度投影, 在分析之前将3维堆栈压缩为2D 图像 (图 2C)。然后手动测量这些数据 (图 2D)。与印度墨水染色细胞的测量不同, 荧光染色使我们能够在实验的所有三重复中, 根据胶囊直径对所有三株菌株进行鉴别。在这里, 我们发现, B18 有最小的胶囊直径和 B52 最大 (p < 0.001 为所有比较, 标准最小二乘法测试以简单的效果, 样品大小 = 50 细胞/应变)。
为了进一步评估两种方法的C. 隐球菌胶囊染色, 三株的测量结果进行了比较。使用两种染色方法确定的胶囊尺寸没有显著差异 (图 3A) (具有简单效果的多变量方差分析, 样本大小 = 50 细胞/应变)。这一结论与一项单独实验的结果一致, H99S 实验室菌株与印度墨水和荧光染料共同染色 (图 3B-c)。然而, 在使用印度墨水的实验之间有更大的变异性, 这证明了一个事实, 即只有三的实验表明, H99S 和 B18 之间的胶囊大小有显著的差异, 与观察到的显著差异相比在所有三种荧光实验 (图3 d-E) 之间的这些菌株之间 (多变量方差分析与简单的效果, 样本大小 = 50 细胞/应变)。
印度墨水染色的图像符合协议中列出的标准 (第5节), 可以使用自动系统进行测量。图 4A中显示的工作流指导用户执行使用设计用于测量C. 隐球菌胶囊的食谱所需的步骤。开发工作流后, 通过使用手动和自动方法, 通过测量三个研究人员的现有图像的缓存来验证它. 在手工制作时, 研究者的测量没有显著的差异, 当使用自动图像分析软件 (图 4B) 进行测量时, 这也是正确的 (方差计算, 样本大小 = 50 细胞/研究员)。但是, 在两种方法 (图 4C) 方差分析、样本大小 = 150 单元/组之间报告的胶囊大小之间存在着微小但显著的差异。通过自动化测量时, 胶囊直径一直略小于手动测量 (0.3 µm, p < 0.001)。另外, 当将图像分析软件用于量化而不是手动方法时, 在比较不同研究人员所做的胶囊尺寸测量时, 标准误差较小, 这暗示了更多使用自动方法进行精确、可重复的测量。为了成功地自动测量, 单元格必须处于焦点, 有一个中型到大的胶囊, 而不是过度聚集 (图 4D)。我们发现, 小胶囊的聚集细胞的聚焦图像不能在软件中自动测量。然而, 我们断言, 这些也可能是挑战, 以准确地测量使用手动方法。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57171/57171fig1.jpg"> 图 1: 印度墨水染色测量C. 隐球菌胶囊.(A)用印度墨水染色法测定的C. 隐球菌胶囊的示意图表示。简而言之, 胶囊表达的诱导使用养分剥夺和接触 CO2, 细胞悬浮在印度墨水和安装在玻璃幻灯片上。染色细胞的图像是由光学显微镜获取的, 使用者选择的领域或使用计算机控制阶段的平铺扫描。图像可以手动分析或处理, 用于自动图像分割和分析。(B) 常见的实验室应变、H99S 和两种临床菌株、B18 和 B52 的图像, 包括胶囊前和后囊诱导。缩放条表示10µm (C)图, 该实验显示了所有三株沾有印度墨水的菌株的胶囊直径, 并手动测量 (错误表示为标准错误 (SE))。统计意义如下: *, p < 0.05;**, p < 0.01;和 **, p < 0.001。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57171/57171fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57171/57171fig2.jpg"> 图 2: 要测量的荧光染色C. 隐球菌胶囊.(A) Calcofluor 白 (CFW) 和 18B7-AF488 共染色法测定的C. 隐球菌胶囊的示意图表示。在胶囊诱导和染色后, 通过激光扫描共聚焦显微镜对细胞进行安装和成像。由于细胞可能在不同的焦平面, z 栈是在每个位置获取。这些被处理以产生最大强度投射, 可用于准确测量每个细胞的最大胶囊直径。(B)常见实验室应变、H99S 和两种临床菌株的图像, B18 和 B52, 后囊诱导. 注意: 缩放条表示所有图像的10µm。(c) 标记为C. 隐球菌单元格的荧光的最大强度投影。(D)图的一个代表性实验显示, 人工测量的胶囊直径所有三株染色的 CFW 和 18B7-AF488 (误差表示为标准误差 (SE)). 统计意义如下: *, p < 0.05;**, p < 0.01;和 **, p < 0.001。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57171/57171fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57171/57171fig3.jpg"> 图 3: 印度墨水与荧光染色结果的比较.(A) 分析与印度墨水或荧光染料染色的三个C. 隐球菌菌株, n = 3 (错误表示为标准错误 (SE))。(B) 单个C. 隐球菌单元格的图像, 其中印有印度墨水和两种荧光染料 (鳞片条代表13µm)。水平线标定胶囊的边缘和垂直线标定细胞体的边缘。(C) 对 H99S 与印度墨水和荧光染料共同染色的代表性实验进行量化 (误差表示为标准误差 (SE))。(D、E)描述印度墨水实验 (D) 和荧光实验 (E) 之间的胶囊直径变异性。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57171/57171fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57171/57171fig4.jpg"> 图 4: 计算分析 C. 隐球菌 胶囊直径来自印度墨水染色细胞的图像.(A) 图像分割和C. 隐球菌胶囊的示意图表示方法使用自动测量技术 (见材料表)。(B) 使用手动 (手动 1-3) 和自动方法 (自动 1-3) 对3个独立调查员进行的胶囊测量进行比较。每个调查员都使用相同的C. 隐球菌图像的缓存, 这两种方法 (错误表示为 StDev)。(C) 手动测量和自动测量的平均值 (错误表示为标准错误 (SE))。 C. 隐球菌的非理想图像 (D) 理想和 (E) 的示例。无法在软件中成功地测量非理想图像。统计意义如下: *, p < 0.05;**, p < 0.01;和 **, p < 0.001。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57171/57171fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Size Matters: Measurement of Capsule Diameter in Cryptococcus neoformans at JoVE.com

Size Matters: Measurement of Capsule Diameter in Cryptococcus neoformans

尺寸事项:新生隐球菌中胶囊直径的测定

多糖胶囊是新生隐球菌的主要毒力因子, 其大小与菌株的毒力密切相关。胶囊直径测量用于表型检测和测量疗效。本文提出了一种标准的胶囊诱导方法, 并比较了两种染色法和测量直径法。

The polysaccharide capsule of Cryptococcus neoformans is the primary virulence factor and one of the most commonly studied aspects of this pathogenic ...

size-matters-measurement-capsule-diameter-cryptococcus

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

Size Matters: Measurement of Capsule Diameter in Cryptococcus neoformans

13.4K 次观看.  Middle Tennessee State University. 多糖胶囊是新生隐球菌的主要毒力因子, 其大小与菌株的毒力密切相关。胶囊直径测量用于表型检测和测量疗效。本文提出了一种标准的胶囊诱导方法, 并比较了两种染色法和测量直径法。

视频: Size Matters: Measurement of Capsule Diameter in Cryptococcus neoformans

Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

Lymphatic vessels comprise a multifunctional transport system that maintains fluid homeostasis, delivers lipids to the central circulation, and acts as a surveillance system for potentially harmful antigens, optimizing mucosal immunity and adaptive immune responses1. Lymph is formed from interstitial fluid that enters blind-ended initial lymphatics, and then is transported against a pressure gradient in larger collecting lymphatics. Each collecting lymphatic is made up of a series of segments called lymphangions, separated by bicuspid valves that prevent backflow. Each lymphangion possesses a contractile cycle that propels lymph against a pressure gradient toward the central circulation2. This phasic contractile pattern is analogous to the cardiac cycle, with systolic and diastolic phases, and with a lower contraction frequency4. In addition, lymphatic smooth muscle generates tone and displays myogenic constriction and dilation in response to increases and decreases in luminal pressure, respectively5. A hybrid of molecular mechanisms that support both the phasic and tonic contractility of lymphatics are thus proposed.
Contraction of smooth muscle is generally regulated by the cytosolic Ca2+ concentration ([Ca2+]i) plus sensitivity to Ca2+, of the contractile elements in response to changes in the environment surrounding the cell6. [Ca2+]i is determined by the combination of the movement of Ca2+ through plasma membrane ligand or voltage gated Ca2+ channels and the release and uptake of Ca2+ from internal stores. Cytosolic Ca2+ binds to calmodulin and activates enzymes such as myosin light chain (MLC) kinase (MLCK), which in turn phosphorylates MLC leading to actin-myosin-mediated contraction8. However, the sensitivity of this pathway to Ca2+ can be regulated by the MLC phosphatase (MLCP)9. MLCP activity is regulated by Rho kinase (ROCK) and the myosin phosphatase inhibitor protein CPI-17.
Here, we present a method to evaluate changes in [Ca2+]i over time in isolated, perfused lymphatics in order to study Ca2+-dependent and Ca2+-sensitizing mechanisms of lymphatic smooth muscle contraction. Using isolated rat mesenteric collecting lymphatics we studied stretch-induced changes in [Ca2+]i and contractile activity. The isolated lymphatic model offers the advantage that pressure, flow, and the chemical composition of the bath solution can be tightly controlled. [Ca2+]i was determined by loading lymphatics with the ratiometric, Ca2+-binding dye Fura-2. These studies will provide a new approach to the broader problem of studying the different molecular mechanisms that regulate phasic contractions versus tonic constriction in lymphatic smooth muscle.

Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

We introduce an approach to evaluate the cytosolic Ca2+ concentration in isolated lymphatics to study Ca2+-dependent and Ca2+-sensitizing mechanisms of lymphatic smooth muscle contraction.

胞内钙的测量 2 +</sup在隔离的收缩淋巴管

Lymphatic vessels comprise a multifunctional transport system that maintains fluid homeostasis, delivers lipids to the central circulation, and acts as a ...

科研

免疫与感染

Measurement of &#947;HV68 Infection in Mice

&gamma;-Herpesviruses (&gamma;-HVs) are notable for their ability to establish latent infections of lymphoid cells1. The narrow host range of human &gamma;-HVs, such as EBV and KSHV, has severely hindered detailed pathogenic studies. Murine &gamma;-herpesvirus 68 (&gamma;HV68) shares extensive genetic and biological similarities with human &gamma;-HVs and is a natural pathogen of murid rodents2. As such, evaluation of &gamma;HV68 infection of mice inbred strains at different stages of viral infection provides an important model for understanding viral lifecycle and pathogenesis during &gamma;-HVs infection.
Upon intranasal inoculation, &gamma;HV68 infection results in acute viremia in the lung that is later resolved into a latent infection of splenocytes and other cells, which may be reactivated throughout the life of the host3,4. In this protocol, we will describe how to use the plaque assay to assess infectious virus titer in the lung homogenates on Vero cell monolayers at the early stage (5 - 7 days) of post-intranasal infection (dpi). While acute infection is largely cleared 2 - 3 weeks postinfection, a latent infection of &gamma;HV68 is established around 14 dpi and maintained later on in the spleen of the mice. Latent infection usually affects a very small population of cells in the infected tissues, whereby the virus stays dormant and shuts off most of its gene expression. Latently-infected splenocytes spontaneously reactivate virus upon explanting into tissue culture, which can be recapitulated by an infectious center (IC) assay to determine the viral latent load. To further estimate the amount of viral genome copies in the acutely and/or latently infected tissues, quantitative real-time PCR (qPCR) is used for its maximal sensitivity and accuracy. The combined analyses of the results of qPCR and plaque assay, and/or IC assay will reveal the spatiotemporal profiles of viral replication and infectivity in vivo.

&#947;-Herpesviruses (&#947;-HVs) establish life-long persistency in their host. Infection of mice with &#947;-HV68 provides a genetically tractable in vivo model for the characterization of the lifecycle/pathogenesis of &#947;HVs. This protocol describes the detection and quantitation of &#947;HV68 infection at acute and latent stages following infection by plaque-forming, infectious center, and qPCR assays.

γHV68感染小鼠的测量

&gamma;-Herpesviruses (&gamma;-HVs) are notable for their ability to establish latent infections of lymphoid cells1. The narrow host range of human ...

Quantitative Measurement of the Immune Response and Sleep in Drosophila

A complex interaction between the immune response and host behavior has been described in a wide range of species. Excess sleep, in particular, is known to occur as a response to infection in mammals 1 and has also recently been described in Drosophila melanogaster2. It is generally accepted that sleep is beneficial to the host during an infection and that it is important for the maintenance of a robust immune system3,4. However, experimental evidence that supports this hypothesis is limited4, and the function of excess sleep during an immune response remains unclear. We have used a multidisciplinary approach to address this complex problem, and have conducted studies in the simple genetic model system, the fruitfly Drosophila melanogaster. We use a standard assay for measuring locomotor behavior and sleep in flies, and demonstrate how this assay is used to measure behavior in flies infected with a pathogenic strain of bacteria. This assay is also useful for monitoring the duration of survival in individual flies during an infection. Additional measures of immune function include the ability of flies to clear an infection and the activation of NF&kappa;B, a key transcription factor that is central to the innate immune response in Drosophila. Both survival outcome and bacterial clearance during infection together are indicators of resistance and tolerance to infection. Resistance refers to the ability of flies to clear an infection, while tolerance is defined as the ability of the host to limit damage from an infection and thereby survive despite high levels of pathogen within the system5. Real-time monitoring of NF&kappa;B activity during infection provides insight into a molecular mechanism of survival during infection. The use of Drosophila in these straightforward assays facilitates the genetic and molecular analyses of sleep and the immune response and how these two complex systems are reciprocally influenced.

Quantitative Measurement of the Immune Response and Sleep in Drosophila

To understand a link between the immune response and behavior, we describe a method to measure locomotor behavior in Drosophila during bacterial infection as well as the ability of flies to mount an immune response by monitoring survival, bacterial load, and real-time activity of a key regulator of innate immunity, NF&kappa;B.

定量测定的免疫反应和睡眠模式<em&gt;果蝇</em

A complex interaction between the immune response and  host behavior has been described in a wide range of species. Excess sleep, in  particular, is known ...

Measurement of Tactile Allodynia in a Murine Model of Bacterial Prostatitis

Uropathogenic Escherichia coli (UPEC) are pathogens that play an important role in urinary tract infections and bacterial prostatitis1. We have recently shown that UPEC have an important role in the initiation of chronic pelvic pain2, a feature of Chronic prostatitis/Chronic pelvic pain syndrome (CP/CPPS)3,4. Infection of the prostate by clinically relevant UPEC can initiate and establish chronic pain through mechanisms that may involve tissue damage and the initiation of mechanisms of autoimmunity5.
A challenge to understanding the pathogenesis of UPEC in the prostate is the relative inaccessibility of the prostate gland to manipulation. We utilized a previously described intraurethral infection method6 to deliver a clinical strain of UPEC into male mice thereby establishing an ascending infection of the prostate. Here, we describe our protocols for standardizing the bacterial inoculum7 as well as the procedure for catheterizing anesthetized male mice for instillation of bacteria.
CP/CPPS is primarily characterized by the presence of tactile allodynia4. Behavior testing was based on the concept of cutaneous hyperalgesia resulting from referred visceral pain8-10. An irritable focus in visceral tissues reduces cutaneous pain thresholds allowing for an exaggerated response to normally non-painful stimuli (allodynia). Application of normal force to the skin result in abnormal responses that tend to increase with the intensity of the underlying visceral pain. We describe methodology in NOD/ShiLtJ mice that utilize von Frey fibers to quantify tactile allodynia over time in response to a single infection with UPEC bacteria.

Infection of the prostate may be a contributing factor in mediating pelvic pain in chronic prostatitis. We describe the procedure for preparation of standardized bacterial inoculum, instillation of bacteria into the urethra of male mice and methodology for measuring tactile allodynia in mice over time.

细菌性前列腺炎小鼠模型的测量触觉异常

Uropathogenic Escherichia coli (UPEC) are pathogens that play an important role in urinary tract infections and bacterial prostatitis1. We have recently ...

Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy

Many aspects of the infection of macrophages by Cryptococcus neoformans have been extensively studied and well defined. However, one particular interaction that is not clearly understood is non-lytic exocytosis. In this process, yeast cells are released into the extracellular space by a poorly understood mechanism that leaves both the macrophage and Cn viable. Here, we describe how to follow a large number of individually infected macrophages for a 24 hr infection period by time-lapsed microscopy. Infected macrophages are housed in a heating chamber with a CO2 atmosphere attached to a microscope that provides the same conditions as a cell-culture incubator. Live digital microscopy can provide information about the dynamic interactions between a host and pathogen that is not available from static images. Being able to visualize each infected cell can provide clues as to how macrophages handle fungal infections, and vice versa. This technique is a powerful tool in studying the dynamics that are behind a complex phenomenon.

Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy

We describe how to visualize macrophage-C. neoformans (Cn) interactions in real time, with specific emphasis on the process of non-lytic exocytosis using digital light microscopy. Using this technique individually infected macrophages can be studied to ascertain various aspects of this phenomenon.

可视化的非溶胞胞吐作用<em&gt;新型隐球菌</em&gt;从巨噬细胞用数码光学显微镜

Many aspects of the infection of macrophages by Cryptococcus neoformans have been extensively studied and well defined. However, one particular ...

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are able to replicate in the deep lung. Phagocytosis of Cryptococcus by macrophages is one of the ways that the disease is able to spread into the central nervous system to cause lethal meningoencephalitis. Therefore, study of the association between Cryptococcus and macrophages is important to understanding the progression of the infection. The present study describes a step-by-step protocol to study macrophage infectivity by C. neoformansin vitro. Using this protocol, the role of host sterols on host-pathogen interactions is studied. Different concentrations of methyl--cyclodextrin (MCD) were used to deplete cholesterol from murine reticulum sarcoma macrophage-like cell line J774A.1. Cholesterol depletion was confirmed and quantified using both a commercially available cholesterol quantification kit and thin layer chromatography. Cholesterol depleted cells were activated using Lipopolysacharide (LPS) and Interferon gamma (IFN&#947;) and infected with antibody-opsonized Cryptococcus neoformans wild-type H99 cells at an effector-to-target ratio of 1:1. Infected cells were monitored after 2 hr of incubation with C. neoformans and their phagocytic index was calculated. Cholesterol depletion resulted in a significant reduction in the phagocytic index. The presented protocols offer a convenient method to mimic the initiation of the infection process in a laboratory environment and study the role of host lipid composition on infectivity.

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

In this article, a protocol for infection of macrophages with Cryptococcus neoformans is described. Also, a method for sterol depletion from the macrophages is explained. These protocols provide a guide to study fungal infections in vitro and examine the role of sterols in such infections.

巨噬细胞胆固醇消耗及其对的吞噬作用<em&gt;新型隐球菌</em

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are ...

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal meningitis worldwide resulting in more than 625,000 deaths per year worldwide.&#160;Although electroporation has been developed for the transformation of plasmids in Cryptococcus, only&#160;biolistic&#160;delivery provides&#160;an effective means to transform&#160;linear&#160;DNA&#160;that can be integrated into the genome by homologous recombination.&#160;
Acetate has been shown to be a major fermentation product during cryptococcal infection, but the significance of this is not yet known.&#160;A bacterial pathway composed of the enzymes xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (Xfp) and acetate kinase (Ack) is one of three potential pathways for acetate production in C. neoformans. Here, we demonstrate the biolistic transformation of a construct, which has the gene encoding Ack fused to the fluorescent tag mCherry, into&#160;C. neoformans. We then confirm integration of the ACK-mCherry fusion into the&#160;ACK&#160;locus.

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

Biolistic transformation is a method used to generate stable integration of DNA into the genome of the opportunistic pathogen Cryptococcus neoformans through homologous recombination. We will demonstrate biolistic transformation of a construct, which has the gene encoding acetate kinase fused to the fluorescent tag mCherry into&#160;C. neoformans.

荧光标记基因的基因枪转化成机会性真菌病原<em&gt;新型隐球菌</em

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal ...

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the viral capsid (CA) core into the cytoplasm. Subsequently, the viral Reverse Transcriptase (RT), as part of a namesake nucleoprotein complex termed the Reverse Transcription Complex (RTC), converts the viral single-stranded RNA genome into a double-stranded DNA copy (vDNA). This leads to the biogenesis of another nucleoprotein complex, termed the pre-integration complex (PIC), composed of the vDNA and associated virus proteins and host factors. The PIC-associated viral integrase (IN) orchestrates the integration of the vDNA into the host chromosomal DNA in a temporally and spatially regulated two-step process. First, the IN processes the 3&#39; ends of the vDNA in the cytoplasm and, second, after the PIC traffics to the nucleus, it mediates integration of the processed vDNA into the chromosomal DNA. The PICs isolated from target cells acutely infected with HIV-1 are functional in vitro, as they are competent to integrate the associated vDNA into an exogenously added heterologous target DNA. Such PIC-based in vitro integration assays have significantly contributed to delineating the mechanistic details of retroviral integration and to discovering IN inhibitors. In this report, we elaborate upon an updated HIV-1 PIC assay that employs a nested real-time quantitative Polymerase Chain Reaction (qPCR)-based strategy for measuring the in vitro integration activity of isolated native PICs.

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

This report describes an in vitro assay for measuring the human immunodeficiency virus type 1 (HIV-1) preintegration complex (PIC)-associated integration activity in the cytoplasmic extracts of acutely infected cells. The integration of PIC-associated HIV-1 DNA into heterologous target DNA is quantified by using a nested real-time polymerase chain reaction strategy.

测量<em&gt;体外</em&gt; HIV-1 Preintegration配合物的整合活动

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the ...

Automated Measurement of Cryptococcal Species Polysaccharide Capsule and Cell Body

The purpose of this technique is to provide a consistent, accurate, and manageable process for large numbers of polysaccharide capsule measurements.
First, a threshold image is generated based on intensity values uniquely calculated for each image. Then, circles are detected based on contrast between the object and background using the well-established Circle Hough Transformation (CHT) algorithm. Finally, the detected cell capsules and bodies are matched according to center coordinates and radius size, and data is exported to the user in a manageable spreadsheet.
The advantages of this technique are simple but significant. First, because these calculations are performed by an algorithm rather than a human both accuracy and reliability are increased. There is no decline in accuracy or reliability regardless of how many samples are analyzed. Second, this approach establishes a potential standard operating procedure for the Cryptococcus field instead of the current situation where capsule measurement varies by lab. Third, given that manual capsule measurements are slow and monotonous, automation allows rapid measurements on large numbers of yeast cells that in turn facilitates high throughput data analysis and increasingly powerful statistics.
The major limitations of this technique come from how the algorithm functions. First, the algorithm will only generate circles. While Cryptococcus cells and their capsules take on a circular morphology, it would be difficult to apply this technique to non-circular object detection. Second, due to how circles are detected the CHT algorithm can detect enormous pseudo-circles based on the outer edges of several clustered circles. However, any misrepresented cell bodies caught within the pseudo-circle can be easily detected and removed from the resulting data sets.
This technique is meant for measuring the circular polysaccharide capsules of Cryptococcus species based on India Ink bright field microscopy; though it could be applied to other contrast based circular object measurements.

Automated Measurement of Cryptococcal Species Polysaccharide Capsule and Cell Body

This technique describes an automated batch image processor designed to measure polysaccharide capsule and body radii. While initially designed for Cryptococcus neoformans capsule measurements the automated image processor can also be applied to other contrast based detection of circular objects.

自动测量隐球菌种多糖胶囊和细胞体

The purpose of this technique is to provide a consistent, accurate, and manageable process for large numbers of polysaccharide capsule measurements.
...

A Method for the Measurement of Salivary Gland Function in Mice

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced saliva production. Similarly, saliva production is severely compromised in patients receiving radiation treatment for head and neck cancers. Rodent models, developed to mimic these clinical conditions, facilitate an understanding of the disease pathogenesis and allow for the development of new therapeutic strategies. Therefore, the ability to accurately, reproducibly, and repeatedly measure salivary gland function in animal models is critical. Building on procedures previously described in the literature, a method was developed that meets these criteria and was used to evaluate salivary gland function in mice. An additional advantage of this new method is that it is easily mastered, and has little inter-operator variation. Salivary gland function is evaluated as the amount (weight or volume) or rate (mL/min) of saliva produced in response to pilocarpine stimulation. The collected saliva is a good source for the analyses of protein content, immunoglobulin concentrations, and other biomolecules.

Salivary gland hypofunction is a frequent consequence of autoimmune disease and radiation therapy. Reproducible evaluation of salivary gland function in mouse models of these diseases is a technical challenge. Here, a simple method for accurate and reproducible measurement of saliva production in mice is described.

小鼠涎腺功能的测定方法

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced ...