我们要感谢安东尼. 鲍文, 他的幻灯片被作为第二个人并排比较, 以及他的幻灯片被用作第三人并排和第二次显微镜比较。

1. 印墨幻灯片的制作
<ol><li>吸管10µL 的隐球菌样本到幻灯片上。任何循环酵母菌株将工作, 但这个实验 H99 是唯一的应变使用。 注: 如果样品直接来自培养基, 用 PBS 或水稀释1:2 可以帮助防止墨汁结块。</li>
	<li>吸管2µL 印度墨水污点的样品和混合通过物理推动吸管尖端的样品和移动, 直到印度墨水出现均匀分布。</li>
	<li>通过将片的左边缘与幻灯片的表面片, 然后在样品上轻轻地、均匀地降低片的另一侧, 在样品上放置一个。</li>
	<li>允许滑动到空气干燥5分钟。</li>
	<li>轻轻地涂上一层指甲油到片边界, 形成一个印章, 并保存印度墨水污点。</li>
</ol>2. 影像幻灯片
<ol><li>将幻灯片放在明亮的野外显微镜下, 用照相机附件和已知的 pixel-to-micron 转换。调整滤镜、目标和对比度, 使细胞胶囊清晰, 细胞体聚焦, 暗色带。 注意: 各种滤镜、目标和对比度设置都可以使用, 但建议使用20x 目标、Ph1 过滤器和2x2 分。</li>
	<li>确保视野是稠密的, 但不是过度的与隐球菌细胞, 与细胞囊和背景清晰的对比, 并适当集中在细胞体可视化为一个黑暗的波段。 注意: 最佳图像的精确单元格数将因所使用的样本和目标而异。重要的方面是确保细胞不聚集或重叠, 细胞的焦平面不明显变化, 并有明显的印度墨水污点清晰可见的背景 (至少25% 的领域)。</li>
	<li>将图像保存到具有明确标题的单个目录中, 因为测量算法将在单个目录中的图像上运行, 并且输出数据将根据图像文件的名称进行组织。</li>
</ol>3. 算法设置
<ol><li>安装 Python 版本2。7</li>
	<li>通过运行 "pip 安装枕头" 和 "pip 安装 openpyxl" 命令安装附加的 python 库。</li>
	<li>按照以下说明安装 MATLAB</li>
	<li>按照 https://www.mathworks.com/help/matlab/matlab_external/install-the-matlab-engine-for-python.html 提供的说明构建 MATLAB python 库。</li>
	<li>下载这份手稿的补充材料 ("QCA", "Analysis2", 和 "TestRun m") 所需的三文件。 注意: 这些文件可以提取到任何位置, 但所有三必须在同一目录中。</li>
</ol>4. 算法的使用
<ol><li>通过双击 QCA 运行应用程序。 注意: 应用程序可能需要几分钟才能开始。"QCA" 文件包含调用 ". m" 文件的程序结构来运行实际算法。</li>
	<li>按照程序中概述的步骤操作。<ol>
<li>输入图像文件的扩展类型, 前面是句点, 用分号 (ex) 分隔。TIF;。jpeg ") 然后单击Enter按钮。</li>
		<li>通过单击选择目录"按钮并选择包含图像的文件夹, 选择图像文件所在的目录。</li>
		<li>通过单击生成图像列表按钮, 在目录中生成图像文件的列表。图像将在右侧的文本框中列出。检查并确保清单的准确性和完整性。</li>
		<li>通过单击选择随机图像"按钮, 从列表中选择一个随机图像以用作预览。 注意: 如果由于图像模式错误而无法打开图像, 该算法将仍然正常工作, 尽管不显示图像。步骤4.2.7 后, 测试图像仍将显示。</li>
		<li>输入显微镜物镜和分设置。如果默认设置与使用的显微镜不匹配, 请选择 "自定义像素转换" 并输入图像文件的 pixel-to-um 转换。选定后, 单击计算转换"按钮, 并确保根据右侧的文本框进行的转换是正确的。 注意: 在图 1中显示的具有代表性的图像是用40x 的放大倍数和2x2 分的选择来计算的。</li>
		<li>用于圆检测的输入算法参数。<ol>
<li>输入最小和最大半径可检测的外舱探测作为最小和最大胶囊半径条目。较小的范围将允许更准确的结果。</li>
			<li>输入最小和最大半径检测为细胞体探测作为最小和最大细胞体半径条目。 注意: 根据源图像, 所有四个条目都应该是以像素为单位表示各自值的数字。</li>
			<li>移动胶囊和细胞体灵敏度滑块来调整算法的灵敏度阈值。低灵敏度将是严格的, 并减少假阳性圆检测, 但也可能检测到更少的真正的圈子。反之, 高灵敏度将提高检测率, 但也可能导致假阳性圆。 注: 有代表性的结果, 最小胶囊半径为 7, 最大胶囊半径为 45, 最小体半径为 4, 最大体半径为 30, 胶囊灵敏度 87, 体灵敏度87。</li>
		</ol>
</li>
		<li>通过单击运行测试按钮来测试随机选择的图像上的参数。结果将显示在程序的上部中心, 替换原始图像。如果结果看起来准确, 建议选择一个额外的随机图像, 以确保选定的参数将适合所有选定的图像。<ol>
<li>最大化检测到的物体和胶囊的数量, 并目视检查这些圆圈是否适合正确显示。检测到的马蹄形内的主体数量将显示在右侧的文本框中。否则, 操作算法参数, 直到结果是准确的。 注: 厚色圆圈只对可视化有帮助。实际产生的测量圆是一个数学曲线计算的小贩的算法。</li>
		</ol>
</li>
		<li>单击开始分析按钮, 在整个映像文件目录中运行检测算法。每个图像将被分析, 程序将显示 "完成" 在文本框的权利, 当所有的图像都被分析。</li>
		<li>单击匹配和清除按钮。这将匹配检测到的细胞体的检测胶囊, 他们驻留在, 并计算真正的胶囊半径减去身体从胶囊。 注意: 如果该算法被用于检测荧光或其他只检测到一个圆圈的情况, 这一步是不必要的。而只需单击仅拉正文或仅拉式胶囊按钮, 即可分别只收集蓝色或绿色圆圈的数据。重要的是要注意, 如果只检索胶囊的数据, 半径将引用的细胞的总半径, 因为不能减去细胞体的半径。</li>
		<li>在映像目录中的 "CleanedOuput. csv" 文件中找到已完成的数据。如果仅选择了一条数据, 则该文件将被标记为 "CleanedBodies" 或 "CleanedCapsules. csv"。</li>
	</ol>
</li>
</ol>

<ol>
	<li>Park, B. J., Wannemuehler, K. A., Marston, B. J., Govender, N., Pappas, P. G., Chiller, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimation+of+the+current+global+burden+of+cryptococcal+meningitis+among+persons+living+with+HIV/AIDS.">Estimation of the current global burden of cryptococcal meningitis among persons living with HIV/AIDS.</a> AIDS. 23 (4), 525-530 (2009).</li><li>Kwon-Chung, K. J., 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=Cryptococcus+neoformans+and+Cryptococcus+gattii,+the+etiologic+agents+of+cryptococcosis.">Cryptococcus neoformans and Cryptococcus gattii, the etiologic agents of cryptococcosis.</a> Cold Spring Harb Perspect Med. 4 (7), 019760 (2014).</li><li>Granger, D. L., Perfect, J. R., Durack, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virulence+of+Cryptococcus+neoformans.+Regulation+of+capsule+synthesis+by+carbon+dioxide.">Virulence of Cryptococcus neoformans. Regulation of capsule synthesis by carbon dioxide.</a> J Clin Invest. 76 (2), 508 (1985).</li><li>Rumbaugh, J., Pool, A., Gainey, L., Forrester, K., Wu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+Cryptococcal+Capsule+in+Pathogenesis+of+Cryptococcal+Meningitis.">The Role of Cryptococcal Capsule in Pathogenesis of Cryptococcal Meningitis.</a> Neurology. 80 (7), 007 (2013).</li><li>Bojarczuk, A., 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=Cryptococcus+neoformans+Intracellular+Proliferation+and+Capsule+Size+Determines+Early+Macrophage+Control+of+Infection.">Cryptococcus neoformans Intracellular Proliferation and Capsule Size Determines Early Macrophage Control of Infection.</a> Sci Rep. 6, (2016).</li><li>Robertson, E. J., 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=Cryptococcus+neoformans+Ex+Vivo+Capsule+Size+Is+Associated+With+Intracranial+Pressure+and+Host+Immune+Response+in+HIV-associated+Cryptococcal+Meningitis.">Cryptococcus neoformans Ex Vivo Capsule Size Is Associated With Intracranial Pressure and Host Immune Response in HIV-associated Cryptococcal Meningitis.</a> J Infect Dis. 209 (1), 74-82 (2014).</li><li>Araujo, G. d. e. S., 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=Capsules+from+Pathogenic+and+Non-Pathogenic+Cryptococcus+spp.+Manifest+Significant+Differences+in+Structure+and+Ability+to+Protect+against+Phagocytic+Cells.">Capsules from Pathogenic and Non-Pathogenic Cryptococcus spp. Manifest Significant Differences in Structure and Ability to Protect against Phagocytic Cells.</a> PLoS One. 7 (1), 29561 (2012).</li><li>Garc&#237;a-Rivera, J., Chang, Y. C., Kwon-Chung, K. J., Casadevall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryptococcus+neoformans+CAP59+(or+Cap59p)+Is+Involved+in+the+Extracellular+Trafficking+of+Capsular+Glucuronoxylomannan.">Cryptococcus neoformans CAP59 (or Cap59p) Is Involved in the Extracellular Trafficking of Capsular Glucuronoxylomannan.</a> Eukaryot Cell. 3 (2), 385-392 (2004).</li><li>Guimar&#227;es, A. J., Frases, S., Cordero, R. J. B., Nimrichter, L., Casadevall, A., Nosanchuk, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryptococcus+neoformans+responds+to+mannitol+by+increasing+capsule+size+in+vitro+and+in+vivo:+Mannitol+impacts+the+structure+of+C.+neoformans+capsule.">Cryptococcus neoformans responds to mannitol by increasing capsule size in vitro and in vivo: Mannitol impacts the structure of C. neoformans capsule.</a> Cell Microbiol. 12 (6), 740-753 (2010).</li><li>Zaragoza, O., Fries, B. C., Casadevall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+Capsule+Growth+in+Cryptococcus+neoformans+by+Mammalian+Serum+and+CO2.">Induction of Capsule Growth in Cryptococcus neoformans by Mammalian Serum and CO2.</a> Infect and Immun. 71 (11), 6155-6164 (2003).</li><li>Rossi, S. 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作者没有利益冲突透露。

这一技术的关键步骤是准备印度墨水幻灯片和获取显微镜图像。虽然该算法已成功地测试了各种幻灯片和图像技术的推荐协议在这篇手稿中描述。该多糖胶囊是基于印度墨水微粒从胶囊的领域排除, 因为这些粒子太大, 渗透到多糖纤维网络。印度油墨的排斥导致一个明亮的圆圈顶部的黑暗背景。该算法根据它们与背景的对比度来检测圆。因此, 大量印墨色斑会导致多糖胶囊与背景的对比度增高, 增?...

新生隐球菌是一种致病性酵母, 在全球范围内发现无所不在, 与人类疾病主要是免疫人群有关。C. 隐球菌最显著的原因是在撒哈拉以南非洲地区因传染性疾病而导致的全年死亡的一个重要因素1。隐球菌感染的主要临床表现是脑炎, 它是在受感染的巨噬细胞 (特洛伊木马方式) 或直接越过血脑屏障之后, 通过中枢神经系统的侵袭而发生的。C. 隐球菌表达了多种致病因子, 包括在人体温度、脲酶活性、黑和多糖胶囊的形成过程中复制的能力2。该多糖胶囊是由重复 glucuronoxylomannan 和 glucoronoxylomannangalactan 聚合物和功能作为保护屏障的因素, 如环境压力和宿主免疫应答2。
虽然隐球菌多糖胶囊大小的大小并没有持续与毒力相关, 但有证据表明它是致病因素2,3,4,5, 6,7。胶囊大小与脑膜炎病理相关6, 可影响巨噬细胞控制新生感染的能力5, 如果缺席则可能导致毒力丧失8。因此, 胶囊大小测量是常见的隐球菌研究, 但没有 fieldwide 标准的方法的胶囊测量。
目前, C. 隐球菌多糖胶囊的测量是基于手工测量的显微图像, 而且图像和测量的精确方法在实验室中各有不同9,10, 11。这种方法的一个直接的关注是, 一些研究需要获得数以千计的个人测量, 这使得保持准确性和可靠性的困难。此外, 即使在公布结果时, 对测量方法的描述也往往不够充分。许多出版物不解释如何获得他们的测量, 使用了什么焦平面, 他们如何确定胶囊的识别阈值, 无论他们使用的半径或直径, 无论是使用一个测量或平均数, 或其他详细.有些出版物仅说明使用的程序的方法, 例如, "Adobe Photoshop CS3 用于测量单元格"11。这种缺乏标准化和报告细节的情况可能会使重现性困难, 如果不是不可能的话。人的视力、电脑亮度、显微镜设置、滑动照明和其他因素之间的差异不仅在个体之间, 而且在样本之间也会有所不同, 而基于像素强度值的比值的计算将保持不变,适用于样品之间。这项技术是在提供一个标准化的, 准确的, 快速的, 简单的技术来测量胶囊大小的领域中, 没有以前。
如前所述, 海隧算法是长期的, 并且脚本自动地检测圈子以前被写了。此方法在其他脚本不足的两个方面有所改进。首先, 仅仅检测圆是不够的, 因为与隐球菌细胞的两个不同的圆圈必须相互关联检测。这种方法专门检测胶囊内的细胞体, 区分两者之间, 并只对相关的身体-胶囊对进行计算。第二, 即使遵循相同的协议, 不同的调查人员最终会得到不同的获取图像。通过允许调查人员控制每个算法参数, 可以调整该工具以匹配广泛的获取方法。不需要标准化的范围、目标、筛选等。
这种技术可以很容易地应用到任何情况下, 调查人员需要检测的圆圈内的图像, 与他们的背景形成对比。两个圆圈比他们的背景更亮, 更暗, 可以检测, 计数, 并用这种技术测量。

<table border="0" cellpadding="0" cellspacing="0" style="width:875px;" width="875">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:213px;">India Ink</td>
			<td style="width:99px;">Becton, Dickinson and Co.</td>
			<td style="width:119px;">261194</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:213px;">Fisherbrand Superfrost Microscope Slides</td>
			<td style="width:99px;">Fisher Scientific</td>
			<td style="width:119px;">12-550-143</td>
			<td>25x75x1</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:213px;">Fisherfinest Premium Cover Glass</td>
			<td style="width:99px;">Fisher Scientific</td>
			<td style="width:119px;">12-548-B</td>
			<td>22x22-1</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:213px;">Sally Hansen HardasNails Xtreme Wear Nail Polish</td>
			<td style="width:99px;">Sally Hansen</td>
			<td style="width:119px;">N/A</td>
			<td>109 invisible</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">SAB Media</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:119px;">S3306</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Cryptotoccus neoformans</td>
			<td style="width:99px;">ATCC</td>
			<td style="width:119px;">208821</td>
			<td>H99 strain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Olympus AX70 Microscope</td>
			<td style="width:99px;">Olympus</td>
			<td style="width:119px;">AX70TRF</td>
			<td>Discontinued ; Bright Field Microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Qimaging Retiga 1300</td>
			<td style="width:99px;">Qimaging</td>
			<td style="width:119px;">N/A</td>
			<td>Discontinued ; Camera Microscope Attachment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">MATLAB</td>
			<td style="width:99px;">MathWorks</td>
			<td style="width:119px;">N/A</td>
			<td>Most recent version recommended</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Python Programming Language</td>
			<td style="width:99px;">Python</td>
			<td style="width:119px;">N/A</td>
			<td>Version 2 necessary ; 2.7 recommended</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Microsoft Excel</td>
			<td style="width:99px;">Microsoft</td>
			<td style="width:119px;">N/A</td>
			<td>Most recent version recommended</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:213px;">Phosphate Buffered Saline (PBS)</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:119px;">P3813</td>
			<td></td>
		</tr>
	</tbody>
</table>

automated measurement of cryptococcal species polysaccharide capsule and cell body

这项技术的目的是为大量的多糖胶囊测量提供一个一致的, 准确的, 可管理的过程。
首先, 根据每个图像的唯一计算的强度值生成阈值图像。然后, 利用完备的圆霍夫变换 (红隧) 算法, 根据对象和背景的对比度来检测圆。最后, 根据中心坐标和半径大小对检测到的细胞胶囊和体进行匹配, 并在可管理的电子表格中将数据导出到用户。
这种技术的优点很简单但意义重大。首先, 由于这些计算是由一个算法而不是一个人来执行的, 所以精确度和可靠性都提高了。 无论分析多少样品, 准确度或可靠性都没有下降。其次, 此方法为 "隐球菌" 字段建立了一个潜在的标准操作过程, 而不是实验室中的胶囊测量变化的当前情况。第三, 由于手动胶囊测量是缓慢和单调的, 自动化允许快速测量大量的酵母细胞, 反过来又有利于高吞吐量数据分析和日益强大的统计。
该技术的主要局限性来自于该算法的功能。首先, 该算法将只生成圆。当隐球菌细胞及其胶囊采用圆形形态时, 很难将这种技术应用于非圆形物体的检测。其次, 由于如何检测圆, 红隧算法可以根据多个簇圆的外边缘检测出巨大的 pseudo-circles。但是, 在 pseudo-circle 中捕获的任何错误的单元格都可以很容易地检测到并从结果数据集中删除。
本技术是以印度油墨的光场显微镜为基础, 测定新生种的圆形多糖胶囊;虽然它可以应用于其他基于对比度的圆形物体测量。

图像首先由印度墨水幻灯片的显微镜, 使用明亮的野外显微镜和相机 (图 1a) 来获得。重要的是要使细胞分离, 并在足够低的密度不压倒的视野, 以及使用足够的污渍, 以创造对比细胞和背景。如协议中所述, 最佳图像的单元格的确切数目将因所使用的样本、显微镜和目标而异。重要的方面是确保细胞不聚集或重叠, 细胞的焦平面不明显变化,...

Watch this Scientific Journal Video about Automated Measurement of Cryptococcal Species Polysaccharide Capsule and Cell Body at JoVE.com

Automated Measurement of Cryptococcal Species Polysaccharide Capsule and Cell Body

自动测量隐球菌种多糖胶囊和细胞体

该技术描述了一种用于测量多糖胶囊和体半径的自动批处理图像处理器。最初设计为新生隐球菌胶囊测量时, 自动图像处理器也可应用于其他基于对比度的圆形物体检测。

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

Research

JoVE Journal

Immunology and Infection

Automated Measurement of Cryptococcal Species Polysaccharide Capsule and Cell Body

7.2K 次观看.  Johns Hopkins Bloomberg School of Public Health. 该技术描述了一种用于测量多糖胶囊和体半径的自动批处理图像处理器。最初设计为新生隐球菌胶囊测量时, 自动图像处理器也可应用于其他基于对比度的圆形物体检测。

视频: Automated Measurement of Cryptococcal Species Polysaccharide Capsule and Cell Body

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see 1,2,3). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy).
Here, we provide a detailed description of a microscopy method used in several recent studies 4, 5, 6, 7, which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ.

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

This protocol provides a step-by-step procedure to monitor single cell behavior of different bacteria in time using automated fluorescence time-lapse microscopy. Furthermore, we provide guidelines how to analyze the microscopy images.

活细胞成像枯草芽孢杆菌肺炎链球菌使用自动定时显微镜

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not ...

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 ...

Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection occurring after allogeneic hematopoietic stem-cell transplantation. Viral-specific T cells from allogeneic donors, including third party donors, can be propagated ex vivo in compliance with current good manufacturing practice (cGMP), employing repeated rounds of antigen-driven stimulation to selectively propagate desired T cells. The identification and isolation of antigen-specific T cells can also be undertaken based upon the cytokine capture system of T cells that have been activated to secrete gamma-interferon (IFN-&#947;). However, widespread human application of the cytokine capture system (CCS) to help restore immunity has been limited as the production process is time-consuming and requires a skilled operator. The development of a second-generation cell enrichment device such as CliniMACS Prodigy now enables investigators to generate viral-specific T cells using an automated, less labor-intensive system. This device separates magnetically labeled cells from unlabeled cells using magnetic activated cell sorting technology to generate clinical-grade products, is engineered as a closed system and can be accessed and operated on the benchtop. We demonstrate the operation of this new automated cell enrichment device to manufacture CMV pp65-specific T cells obtained from a steady-state apheresis product obtained from a CMV seropositive donor. These isolated T cells can then be directly infused into a patient under institutional and federal regulatory supervision. All the bio-processing steps including removal of red blood cells, stimulation of T cells, separation of antigen-specific T cells, purification, and washing are fully automated. Devices such as this raise the possibility that T cells for human application can be manufactured outside of dedicated good manufacturing practice (GMP) facilities and instead be produced in blood banking facilities where staff can supervise automated protocols to produce multiple products.

The goal of this protocol is to manufacture pathogen-specific clinical-grade T cells using a bench-top, automated, second generation cell enrichment device that incorporates a closed cytokine capture system and does not require dedicated staff or use of a GMP facility. The cytomegalovirus pp65-specific-T cells generated can be directly administered to patients.

巨细胞病毒特异性的自动化细胞富集的T细胞用于使用细胞因子捕获系统的临床应用

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection ...

Highly Sensitive Assay for Measurement of Arenavirus-cell Attachment

Arenaviruses are a family of enveloped RNA viruses that cause severe human disease. The first step in the arenavirus life cycle is attachment of viral particles to host cells. While virus-cell attachment can be measured through the use of virions labeled with biotin, radioactive isotopes, or fluorescent dyes, these approaches typically require high multiplicities of infection (MOI) to enable detection of bound virus. We describe a quantitative (q)RT-PCR-based assay that measures Junin virus strain Candid 1 attachment via quantitation of virion-packaged viral genomic RNA. This assay has several advantages including its extreme sensitivity and ability to measure attachment over a large dynamic range of MOIs without the need to purify or label input virus. Importantly, this approach can be easily tailored for use with other viruses through the use of virus-specific qRT-PCR reagents. Further, this assay can be modified to permit measurement of particle endocytosis and genome uncoating. In conclusion, we describe a simple, yet robust assay for highly sensitive measurement of arenavirus-cell attachment.

The first step in the arenavirus life cycle is attachment of viral particles to host cells. We report a quantitative (q)RT-PCR-based assay for ultrasensitive detection and quantitation of arenavirus attachment events.

高灵敏检测方法的沙粒，细胞附着的测量

Arenaviruses are a family of enveloped RNA viruses that cause severe human disease. The first step in the arenavirus life cycle is attachment of viral ...

Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal of immunosuppression. The mixed leukocyte reaction (MLR), limiting dilution assays, and trans-vivo delayed-type hypersensitivity (DTH) assay have all been applied to this question, but these methods have limited predictive ability and/or significant practical limitations that reduce their usefulness. Imaging flow cytometry is a technique that combines the multiparametric quantitative powers of flow cytometry with the imaging capabilities of fluorescent microscopy. We recently made use of an imaging flow cytometry approach to define the proportion of recipient T cells capable of forming mature immune synapses with donor antigen-presenting cells (APCs). Using a well-characterized mouse heart transplant model, we have shown that the frequency of in vitro immune synapses among T-APC membrane contact events strongly predicted allograft outcome in rejection, tolerance, and a situation where transplant survival depends on induced regulatory T cells. The frequency of T-APC contacts increased with T cells from mice during acute rejection and decreased with T cells from mice rendered unresponsive to alloantigen. The addition of regulatory T cells to the in vitro system reduced prolonged T-APC contacts. Critically, this effect was also seen with human polyclonally expanded, naturally occurring regulatory T cells, which are known to control the rejection of human tissues in humanized mouse models. Further development of this approach may allow for a deeper characterization of the alloreactive T-cell compartment in transplant recipients. In the future, further development and evaluation of this method using human cells may form the basis for assays used to select patients for immunosuppression minimization, and it can be used to measure the impact of tolerogenic therapies in the clinic.

This paper describes a method for measuring alloreactivity in a mixed population of T cells using imaging flow cytometry.

使用成像流式细胞仪T细胞同种异体反应性测定

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal ...

Cell-free Biochemical Fluorometric Enzymatic Assay for High-throughput Measurement of Lipid Peroxidation in High Density Lipoprotein

Low high-density lipoprotein cholesterol (HDL-C) levels are one of the most powerful independent negative predictors of atherosclerotic cardiovascular disease (CVD). The structure and function of HDL rather than HDL-C may more accurately predict atherosclerosis. Several HDL protein and lipid compositional changes that impair HDL function occur in inflammatory states such as atherosclerosis. HDL function is usually determined by cell based assays such as cholesterol efflux assay but these assays have numerous drawbacks lack of standardization. Cell-free assays may give more robust measures of HDL function compared to cell-based assays. HDL oxidation impairs HDL function. HDL has a major role in lipid peroxide transport and high amount of lipid peroxides is related to abnormal HDL function. Lipid-probe interactions should be considered when interpreting the results of non-enzymatic fluorescence assays for measuring the lipid oxidative state. This motivated us to develop a cell-free biochemical enzymatic method to assess HDL lipid peroxide content (HDLox) that contributes to HDL dysfunction. This method is based on the enzyme horseradish peroxidase (HRP) and the fluorochrome Amplex Red that can quantify (without cholesterol oxidase) the lipid peroxide content per mg of HDL-C. Here a protocol is describedfor determination of HDL-lipid peroxidation using the fluorochrome reagent. Assay variability can be reduced by strict standardization of experimental conditions. Higher HDLox values are associated with reduced HDL antioxidant function. The readout of this assay is associated with readouts of validated cell-based assays, surrogate measures of cardiovascular disease, systemic inflammation, immune dysfunction, and associated cardiovascular and metabolic risk phenotypes. This technical approach is a robust method to assess HDL function in human disease where systemic inflammation, oxidative stress and oxidized lipids have a key role (such as atherosclerosis).

We describe here a fluorometric cell-free biochemical assay for determination of HDL-lipid peroxidation. This rapid and reproducible assay can be used to determine HDL function in large scale studies and can contribute to our understanding of HDL function in human disease.

无细胞生化荧光酶法测定高密度脂蛋白脂质过氧化的高通量测定

Low high-density lipoprotein cholesterol (HDL-C) levels are one of the most powerful independent negative predictors of atherosclerotic cardiovascular ...

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. albicans is its ability to form biofilms, structured communities of cells attached to biotic and abiotic surfaces. C. albicans biofilms can form on host tissues, such as mucosal layers, and on medical devices, such as catheters, pacemakers, dentures, and joint prostheses. Biofilms pose significant clinical challenges because they are highly resistant to physical and chemical perturbations, and can act as reservoirs to seed disseminated infections. Various in vitro assays have been utilized to study C. albicans biofilm formation, such as microtiter plate assays, dry weight measurements, cell viability assays, and confocal scanning laser microscopy. All of these assays are single end-point assays, where biofilm formation is assessed at a specific time point. Here, we describe a protocol to study biofilm formation in real-time using an automated microfluidic device under laminar flow conditions. This method allows for the observation of biofilm formation as the biofilm develops over time, using customizable conditions that mimic those of the host, such as those encountered in vascular catheters. This protocol can be used to assess the biofilm defects of genetic mutants as well as the inhibitory effects of antimicrobial agents on biofilm development in real-time.

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

This protocol describes the use of a customizable automated microfluidic device to visualize biofilm formation in Candida albicans under host physiological conditions.

全自动微流控装置在白色念珠菌生物膜形成中的可视化

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. ...

Size Matters: Measurement of Capsule Diameter in Cryptococcus neoformans

The polysaccharide capsule of Cryptococcus neoformans is the primary virulence factor and one of the most commonly studied aspects of this pathogenic yeast. Capsule size can vary widely between strains, has the ability to grow rapidly when introduced to stressful or low nutrient conditions, and has been positively correlated with strain virulence. For these reasons, the size of the capsule is of great interest to C. neoformans researchers. The growth of the C. neoformans capsule is induced during phenotypic testing to help understand the effects of different treatments on the yeast or size differences between strains. Here we describe one of the standard methods of capsule induction and compare two accepted methods of staining and measuring capsule diameter: (i) India ink, a negative stain, used in conjunction with conventional light microscopy and (ii) co-staining with fluorescent dyes of both the cell wall and capsule followed by confocal microscopy. Finally, we show how measurement of capsule diameter from India ink-stained samples can be automated using computational image analysis.

Size Matters: Measurement of Capsule Diameter in Cryptococcus neoformans

The polysaccharide capsule is the primary virulence factor in Cryptococcus neoformans, and its size correlates with strain virulence. Capsule diameter measurements are used in phenotypic testing and to gauge therapeutic efficacy. Here a standard method of capsule induction is presented, and two methods of staining and measuring diameter are compared.

尺寸事项:新生隐球菌中胶囊直径的测定

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

Mouse Body Temperature Measurement Using Infrared Thermometer During Passive Systemic Anaphylaxis and Food Allergy Evaluation

Mouse body temperature measurement is of paramount importance for investigating allergies and anaphylactic symptoms. Rectal probes for temperature readings is common, and they have been proven to be accurate and invaluable in this regard. However, this method of temperature measurement requires the mice to be anesthetized in order to insert the probe without injury to the animal. This limits the ability to observe other phenotypes of the mouse simultaneously. In order to investigate other phenotypes while measuring temperatures, rectal probes are not ideal, and another method is desired. Here, we introduce a noninvasive method of temperature measurement that foregoes the requirement for mouse anesthesia while maintaining equal reliability to rectal probes in measuring body temperature. We use an infrared thermometer that detects body surface temperatures at ranges between 2 and 150 mm. This method of body temperature measurement is successful in reliably replicating temperature change trends during passive system anaphylaxis experiments in mice. We show that body surface temperatures are about 2.0 &#176;C lower than rectal probe measurements, but the degree of temperature drop follows the same trend. Furthermore, we use the same technique to observe mice in a food allergy model to evaluate temperature and activity levels simultaneously.

Here we present a new method to accurately measure body temperature differences in passive systemic anaphylaxis (PSA) and food allergy mouse models using an infrared thermometer. This procedure has been accurately duplicated in previous PSA results.

红外测温仪在被动系统过敏反应和食品过敏评价中的应用

Mouse body temperature measurement is of paramount importance for investigating allergies and anaphylactic symptoms. Rectal probes for temperature ...

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

The generation of reactive oxygen species (ROS) is a hallmark of inflammatory processes, but in excess, oxidative stress is widely implicated in various pathologies such as cancer, atherosclerosis and diabetes. We have previously shown that dysfunction of the Nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/ Kelch-like erythroid cell-derived protein 1 (Keap1) signaling pathway leads to extreme ROS imbalance during cutaneous wound healing in diabetes. Since ROS levels are an important indicator of progression of wound healing, specific and accurate quantification techniques are valuable. Several in vitro assays to measure ROS in cells and tissues have been described; however, they only provide a single cumulative measurement per sample. More recently, the development of protein-based indicators and imaging modalities have allowed for unique spatiotemporal analyses. L-012 (C13H8ClN4NaO2) is a luminol derivative that can be used for both in vivo and in vitro chemiluminescent detection of ROS generated by NAPDH oxidase. L-012 emits a stronger signal than other fluorescent probes and has been shown to be both sensitive and reliable for detecting ROS. The time lapse applicability of L-012-facilitated imaging provides valuable information about inflammatory processes while reducing the need for sacrifice and overall reducing the number of study animals. Here, we describe a protocol utilizing L-012-facilitated in vivo imaging to quantify oxidative stress in a model of excisional wound healing using diabetic mice with locally dysfunctional Nrf2/Keap1.

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

We describe a non-invasive in vivo imaging protocol that is streamlined and cost-effective, utilizing L-012, a chemiluminescent luminol-analog, to visualize and quantify reactive oxygen species (ROS) generated in a mouse excisional wound model.

在 vivo小鼠创面模型中活性氧的成像

The generation of reactive oxygen species (ROS) is a hallmark of inflammatory processes, but in excess, oxidative stress is widely implicated in various ...