这项工作得到了NSF DBI生物整合研究所（BII）资助（第2119968号;PI-Ceballos）和阿肯色州INBRE计划，由国家普通医学科学研究所（NIGMS）资助，P20 GM103429来自美国国立卫生研究院。作者还感谢瓦奇塔浸会大学帕特森暑期本科生研究项目的支持。

虽然该协议是为Gordonia terrae编写的，但它也已成功用于Gordonia lacunae，Gordonia rubripertincta和Gordonia westfalica。
1. 细菌制备
<ol><li>在生物安全柜中，使用良好的微生物学规范17 将戈 登氏 菌 CAG3 的单个菌落接种到 1 L 无菌挡板烧瓶中，其中装有 200 mL 蛋白胨酵母钙肉汤18（PYCa），含有 0.01 mg/mL 环己酰亚胺（参见 材料表）。</li>
	<li>将烧瓶在30°C下以250rpm振荡孵育，直到培养物饱和或3天7。</li>
	<li>在 PYCa 肉汤中连续稀释饱和的 G. terrae 培养物，并在 PYCa 平板19，20 上铺平板 100 μL 10−4、10−5 和 10−6 稀释液。将未稀释的饱和培养物在4°C下冷藏。</li>
	<li>将倒置的展开板在30°C下孵育3天。</li>
	<li>孵育后，确定一个“可数板”，即具有20-200个菌落的板。计算该板上的菌落数，并计算每毫升的菌落形成单位（cfu/mL）19，20。</li>
	<li>用 PYCa 肉汤将饱和培养物稀释至 4.0 x 106 cfu/mL 细菌。</li>
</ol>2. 噬菌体制备
注意：报告的代表性结果是用 Gordonia 噬菌体DelRio 21获得的，DelRio21是在 G. terrae上分离的温带噬菌体。这些方法也已成功与其他 戈登菌 体一起使用。
<ol><li>从分离的噬菌体开始，将噬菌体样品在噬菌体缓冲液7中连续稀释至1 x 10−8稀释。通过用 10 μL 的每种噬菌体稀释液感染 0.3 mL 饱和 G. terrae 培养物来进行双层琼脂噬菌体滴度测定7。室温孵育5-10分钟后，将细菌-噬菌体混合物与3mL PYCa顶级琼脂混合，倒入PYCa琼脂平板上。</li>
	<li>将板在30°C下倒置孵育3天或直到形成斑块7。</li>
	<li>孵育后，确定一个“可数板”，即具有20-200块斑块的板。计算该板上的斑块数量，并计算每毫升的斑块形成单位（pfu/mL）7。</li>
</ol>3. 微孔板制备
注意：此方法应使用平底96孔微孔板（参见 材料表）。所有板的制备和装载必须在生物安全柜中完成，并且应使用良好的微生物学规范17。
<ol><li>通过混合 100 μL Triton-X 100、40 mL 100% 异丙醇和 160 mL 去离子水制备防雾盖涂层溶液22.搅拌混合，并在室温下储存。</li>
	<li>在生物安全柜中，将 6 mL 的盖子涂层溶液添加到无菌 96 孔微孔板盖的内表面，握住盖子的边缘并旋转以确保其被溶液覆盖。让溶液在盖子上静置20秒，然后将溶液倒出，并将盖子倒在高压灭菌的纸巾上以一定角度，直到盖子完全干燥，这通常需要35-45分钟。小心握住盖子的边缘。</li>
	<li>为每个 96 孔微孔板准备 20 mL 0.1% 琼脂糖水溶液，微波融化琼脂糖。</li>
	<li>琼脂糖冷却至50-60°C后，将100μL的0.1%琼脂糖移液到板孔之间的所有空间中，将200μL琼脂糖移液到A行和H行以及第1列，第2列，第11列和第12列23 的孔中（图1）。</li>
</ol>4.用细菌和噬菌体装载板
注意：所有板的制备和装载必须在生物安全柜中完成，并且应使用良好的微生物规范17 。
<ol><li>96 孔板每孔中将包含 75 μL 的 2.0 x 106 cfu/mL 细菌9。将 4.0 x 106 cfu/mL 细菌培养物与 2x PYCa 肉汤 1：1 稀释至 2.0 x 106 cfu/mL。每 96 孔板制备 5 mL。</li>
	<li>使用噬菌体缓冲液7 稀释噬菌体裂解物，制成 2.0 x 106 pfu/mL、2.0 x 105 pfu/mL 和 2.0 x 104 pfu/mL 浓度各 1 mL。这将允许微孔板9 内的感染多重性 （MOI） 为 1、0.1 和 0.01。</li>
	<li>要加载微孔板，取用防雾溶液和琼脂糖制备的板，并将 75 μL 的 2.0 x 106 cfu /mL 细菌移液到 3-10 列中，如图 1 所示。</li>
	<li>如图 1所示，向第3列和第4列中加入75 μL无菌噬菌体缓冲液，作为无噬菌体阳性对照，并在每次加入后上下移液混合。向第 5 列和第 6 列中加入 75 μL 2.0 x 106 pfu/mL 噬菌体，向第 7 列和第 8 列中加入 75 μL 2.0 x 105 pfu/mL 噬菌体，向第 9 列和第 10 列中加入 75 μL 2.0 x 104 pfu /mL 噬菌体，每次加入后上下移液混合。</li>
	<li>用 0.5 英寸的标签胶带将板的两个短边都粘住，以部分密封板，同时允许气体交换。</li>
</ol>5. 孵育和吸光度测量
<ol><li>一旦板装载细菌和噬菌体，将它们以250rpm的速度放在微孔板振荡器上，并在30°C下孵育。</li>
	<li>孵育板96小时，从第16小时开始每8小时在酶标仪上以600nm处进行光密度测量。在测量之间将板放回摇床。 注意：从第0小时开始评估4，h 6 h，8 h和12 h的测量周期，并确定从感染后16小时开始的8小时采样周期最好地捕获 了Gordonia-噬菌体相互作用。</li>
	<li>在整个实验过程中监测盖子是否有冷凝。如果观察到冷凝，请根据步骤3.2用另一个涂覆的盖子更换盖子。</li>
	<li>按照协议第6节生成对照和感染的生长曲线。</li>
</ol>6. 数据分析
<ol><li>使用电子表格程序按照 Stacy-Ceballos-Index GitHub 存储库 （https://github.com/eichristenson/Stacy-Ceballos-Index） 中的电子表格布局计算每种噬菌体稀释液的平均值和标准偏差。</li>
	<li>创建对照和感染生长曲线，并使用 R（请参阅材料表）和 DescTools 24、dplyr 25、ggplot226 和 readxl 27 软件包，并按照 Stacy-Ceballos-Index GitHub 存储库 （https://github.com/eichristenson/Stacy-Ceballos-Index） 中的 R 脚本计算感染指标。<ol><li>AUCcon 是未感染对照曲线下的面积，而 AUCinf 是感染曲线16 下的面积。计算AUC，然后使用以下公式根据曲线下面积值PIAUC16计算生长抑制百分比： （1 - [AUCinf/AUCcon]） × 100</li>
		<li>每条曲线上的水平虚线显示生长峰值，未感染的生长峰标记为N渐近线（con）， 感染的生长峰标记为N渐近线（inf）。 确定 N渐近 线值，然后使用以下公式根据这些峰值生长值 PImax16 计算生长抑制百分比： （1 - [N 渐近线（inf）/ N渐近线（con）]） × 100</li>
		<li>根据 PIAUC 和 PI最大值计算 Stacy-Ceballos 指数 I SC 16，如下所示： （PIAUC ×PI 最大值）0，5 通过整合 Stacy-Ceballos 指数随时间变化来计算相对毒力16.</li>
	</ol></li>
</ol>

使用适应性光密度测定法表征放线杆菌噬菌体感染

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作者没有什么可透露的。

这种基于光密度的微孔板方法允许研究噬菌体宿主范围和感染动力学11，并显示了Stacy-Ceballos指数16作为噬菌体毒力的衡量标准。虽然该方法可以与任何噬菌体宿主系统一起使用，但它专门设计用于适应快速微孔板生长测定9，10，11，以用于生长较慢的细菌，如放线菌。快速微孔板测定不能用于缓慢生长的细菌，除非进行修改以解决蒸发和盖子冷凝。该方法描述了这些必要的修改，并首次证明了使用Stacy-Ceballos指数和相关指标16来描述噬菌体感染。
在多天 96 孔板生长曲线测定中，蒸发可能是一项重大挑战;该方法通过将琼脂糖添加到边界孔和孔之间的空间来解决此问题。琼脂糖边缘与防雾盖处理22相结合，在微孔板内提供必要的湿度，并允许可靠的光密度测量。在不增加湿度的情况下，在所需的漫长孵育期内发生大量的边缘效应蒸发23 ，导致人为的高光密度读数。防雾盖处理是必要的修改，因为盖子冷凝也会人为地提高光密度值。在孵育期间摇动平板是推荐的修改，因为放线菌细菌在生长过程中可能会聚集，从而人为地提供高光密度值并有效降低感染的多重性。
在表征感染动力学的实验中，细菌与噬菌体的比例至关重要，因为必须有足够的噬菌体来显示感染效果，但不能太多，以至于宿主细菌种群立即崩溃9或溶原频率显着增加28。在这种方法中，发现对获得一致结果最有效的比率是MOI为1，但MOI为0.1和0.01时也获得了可用结果。实施该方法时，建议选择一种细菌浓度，并在0.01-19，10，11的MOI范围内测试多种噬菌体浓度。
这里描述的这种技术允许在高通量微孔板测定中评估噬菌体 - 宿主相互作用中生长缓慢的细菌，而不是在每个测量间隔29从较大的培养瓶中子采样。此外，通过演示如何适应微孔板生长测定9，10，11，该技术增加了其他基于微孔板的噬菌体测定对生长较慢的细菌的效用，包括噬菌体表征5，6，12和进化研究30，31。最后，该方法演示了使用Stacy-Ceballos指数16来描述噬菌体感染。该指标最初是使用来自古菌病毒模型系统的数据开发的，并根据光密度值计算得出，因此在不同的病毒系统中具有广泛的实用性。

噬菌体或噬菌体是感染细菌的病毒。它们是地球上数量最多的生物实体1，人们普遍认为噬菌体会影响细菌进化和生态系统过程2，3，4。有几种方法可以描述、测量和分析噬菌体宿主范围8和感染动力学5，6，包括基于琼脂的方法，例如双层琼脂方法7和基于光密度的微孔板方法8，9，10，11，12.每种方法都有自己的优点和缺点。由于其效率，使用双层琼脂法的铺板测试是宿主范围测定的“金标准”，但这种方法是时间和劳动密集型的9。快速微孔板方法可在 24 小时或更短的时间内返回结果，对于快速生长的细菌宿主（例如大肠杆菌9，10，11，12）提供出色的结果，但太短而无法显示生长较慢的细菌宿主（如放线菌7，13，14，15）中的噬菌体感染进展。
专为快速生长的细菌设计的基于光密度的微孔板测定无法运行数天来表征生长缓慢的宿主细菌中的感染动力学，而不会发生蒸发并产生人为的高细菌密度。因此，获得有关缓慢生长细菌物种的噬菌体感染动态的可比高通量数据需要适用于这些细菌的专门技术。
这里介绍的微孔板方法减少了蒸发，从而允许缓慢生长的细菌与噬菌体共培养96小时，并能够研究噬菌体感染动力学和宿主范围。该方法还展示了Stacy-Ceballos指数16，这是一种基于光密度的指标，可以在不同的宿主病毒系统之间进行毒力比较。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agarose</td>
			<td>Omni-Pur</td>
			<td>2090</td>
			<td>for filling border wells of microplate&#160;</td>
		</tr>
		<tr>
			<td>Costar 96 Well Lid Low Evaporation Corner Notch</td>
			<td>Corning</td>
			<td>3931</td>
			<td>replacement microplate lid</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Fisher Chemical</td>
			<td>A461-4</td>
			<td>for lid coating</td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>Tecan Spark 20M</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate Shaker with 4-Place Platform</td>
			<td>Thermo Fisher Scientific</td>
			<td>88-861-023</td>
			<td>to shake plates during incubation</td>
		</tr>
		<tr>
			<td>Non-Tissue Culture-Treated Plate 96 well</td>
			<td>Falcon (a Corning Brand)</td>
			<td>351172</td>
			<td>microplate for growth curve assay</td>
		</tr>
		<tr>
			<td>Peptone yeast calcium (PYCa) agar</td>
			<td>Homemade</td>
			<td></td>
			<td>1 g peptone 
			15 g yeast extract 
			15 g agar 
			990 mL dd H2O 
			4.5 mL 1 M CaCl2 
			2.5 mL 40% dextrose 
			1 mL 10 mg/mL cycloheximide</td>
		</tr>
		<tr>
			<td>Peptone yeast calcium (PYCa) broth</td>
			<td>Homemade, from Reference 16</td>
			<td></td>
			<td>1 g peptone 
			15 g yeast extract 
			990 mL dd H2O 
			4.5 ml 1 M CaCl2 
			2.5 mL 40% dextrose 
			1 mL 10 mg/mL cycloheximide</td>
		</tr>
		<tr>
			<td>Peptone yeast calcium (PYCa) top agar</td>
			<td>Homemade</td>
			<td></td>
			<td>1 g peptone 
			15 g yeast extract 
			4 g agar 
			990 mL dd H2O 
			4.5 mL 1M CaCl2 
			2.5 mL 40% dextrose</td>
		</tr>
		<tr>
			<td>Petri plates</td>
			<td>Thermo Fisher Scientific</td>
			<td>FB0875713</td>
			<td>for determination of bacterial concentration and phage titer assay</td>
		</tr>
		<tr>
			<td>Phage Buffer</td>
			<td>Homemade, from Reference 7</td>
			<td></td>
			<td>10 mL 1 M Tris, pH 7.5 
			10 mL 1 M MgSO4 
			4 g NaCl 
			980 ml dd H2O</td>
		</tr>
		<tr>
			<td>R software</td>
			<td>https://www.r-project.org/</td>
			<td>version 4.3.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Disposable PETG Flask Baffled Bottom w/Vented Closure</td>
			<td>Thermo Fisher Scientific</td>
			<td>4116-1000</td>
			<td>for bacterial culture</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma Aldrich</td>
			<td>9036-19-5</td>
			<td>for lid coating</td>
		</tr>
	</tbody>
</table>

an adapted optical density-based microplate assay for characterizing actinobacteriophage infection

噬菌体是自然环境的关键部分，它们具有塑造细菌种群的强大能力。为了了解单个噬菌体如何与缓慢生长的细菌宿主（如放线菌）相互作用，需要一种简单可靠的方法来量化噬菌体存在下的长期细菌生长。分光光度酶标仪可实现高通量重复测量，但长时间孵育小体积可能会带来技术挑战。该程序适应标准的96孔微孔板，以允许噬菌体和细菌的共同培养而无需子采样96小时，每8小时使用分光光度法吸光度值记录细菌生长。使用R分析这些光密度值以产生感染指标，包括生长抑制百分比，相对毒力和Stacy-Ceballos指数。这里概述的方法提供了一种有效的方法来进行和分析延长持续时间的微孔板生长曲线实验，并包括减少蒸发和盖冷凝的修改。这些协议有助于基于微孔板的缓慢生长细菌宿主与其噬菌体之间相互作用的测定。

如果所得生长曲线显示阳性对照细菌种群随时间增加且吸光度没有突然波动，则实验成功。在MOI为1的情况下，有和没有生产性噬菌体感染的成功实验示例分别显示在图 2 和 图3中。 MOI 为 0.01 的生产性感染如图 4 所示。在所有三个图中都可以看到的阳性对照生长模式（绿色曲线）表明细菌正在生长，它们在生长过程中没有结块，并且不存在污染物。结块和污染表现为单个时间点异常高的吸光度。标准偏差通常会随着实验的时间过程而增加;然而，对照曲线和感染曲线之间的急剧增加或标准偏差重叠可能表明一个或多个孔中存在污染或结块。
描述生产性噬菌体感染的生长曲线（ 如图2所示）显示，添加噬菌体的孔中的细菌吸光度随时间推移而降低。如果细菌超出噬菌体的宿主范围，则不会看到细菌密度的降低，如图 3所示。
显示了所有代表性实验的感染指标，图2和图4中的生产性感染的I SC相对较大，图3中没有有效感染宿主细菌的噬菌体的ISC非常小。 
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65482/65482fig01.jpg"> 图1：微孔板布局。 灰色区域填充0.1%琼脂糖。第 3 列和第 4 列中的空白孔是仅含有噬菌体缓冲液和 2.0 x 106 cfu/mL 细菌的无噬菌体阳性对照孔。虚线孔含有噬菌体和 2.0 x 106 cfu/mL 细菌;第 5 列和第 6 列中的大点表示 MOI 为 1，2.0 x 106 pfu/mL 噬菌体;第 7 列和第 8 列中的培养基点表示 2.0 x 105 pfu/mL 噬菌体的 MOI 为 0.1;第 9 列和第 10 列中的小点表示 MOI 为 0.01，噬菌体为 2.0 x 104 pfu/mL。 <a href="https://www.jove.com/files/ftp_upload/65482/65482fig01large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65482/65482fig02.jpg"> 图 2：使用MOI为1的噬菌体进行成功的生长曲线实验，该噬菌体可有效感染宿主细菌。 显示未感染细菌（绿色）和添加噬菌体的细菌（蓝色）的平均吸光度值（±标准偏差）。缩写：AUC = 曲线下的面积;PIAUC = 根据曲线下面积计算的生长抑制百分比;N渐近 线=峰值生长值;PImax = 根据峰值生长值计算的生长抑制百分比;ISC = Stacy-Ceballos 指数。这张图代表了温带 戈登尼亚 噬菌体DelRio感染G . terrae，它是被分离的细菌。 <a href="https://www.jove.com/files/ftp_upload/65482/65482fig02large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65482/65482fig03.jpg"> 图 3：使用MOI为1的噬菌体进行成功的生长曲线实验，该噬菌体不能有效感染宿主细菌。 显示未感染细菌（绿色）和添加噬菌体的细菌（蓝色）的平均吸光度值（±标准偏差）。缩写：AUC = 曲线下的面积;PIAUC = 根据曲线下面积计算的生长抑制百分比;N渐近 线=峰值生长值;PImax = 根据峰值生长值计算的生长抑制百分比;ISC = Stacy-Ceballos 指数。该图代表DelRio的 G. rubripertincta 感染。 <a href="https://www.jove.com/files/ftp_upload/65482/65482fig03large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65482/65482fig04.jpg"> 图 4：使用 MOI 为 0.01 的噬菌体进行成功的生长曲线实验。 显示未感染细菌（绿色）和添加噬菌体的细菌（蓝色）的平均吸光度值（±标准偏差）。缩写：AUC = 曲线下的面积;PIAUC = 根据曲线下面积计算的生长抑制百分比;N渐近 线=峰值生长值;PImax = 根据峰值生长值计算的生长抑制百分比;ISC = Stacy-Ceballos 指数。这张图代表DelRio的 G. terrae 感染。 <a href="https://www.jove.com/files/ftp_upload/65482/65482fig04large.jpg" target="_blank">请点击此处查看此图的大图。</a>

Watch this Scientific Journal Video about An Adapted Optical Density-Based Microplate Assay for Characterizing Actinobacteriophage Infection at JoVE.com

作者聚焦：一种用于表征放线菌噬菌体感染的基于光密度的微孔板检测  

描述了一种基于光密度的微孔板方法，用于量化噬菌体存在下的长期细菌生长，适用于放线菌和其他生长缓慢的细菌。此方法包括减少蒸发和盖冷凝的修改，以及用于计算病毒感染指标的 R 代码，包括曲线下面积、生长最大值和相对毒力。

一种适用于表征放线菌噬菌体感染的基于光密度的微孔板测定

Bacteriophages are a key part of natural environments, and they have a powerful ability to shape bacterial populations. To understand how individual ...

最终，我们对噬菌体 - 宿主相互作用感兴趣。该协议能够测量生长缓慢的细菌中的噬菌体感染，生成描述性的感染指标。该方法有助于研究生长缓慢的放线菌细菌中的噬菌体-宿主协同进化。快速微孔板方法对快速生长的细菌宿主提供了极好的结果，但太短，无法显示生长较慢的细菌宿主（如放线菌）中的细菌噬菌体感染进展。我们的方法适用于生长缓慢的细菌，允许细菌和噬菌体在表征噬菌体感染动态所需的多天内共培养。我们的协议允许评估噬菌体 - 宿主相互作用以评估微孔板中生长缓慢的细菌，而不是要求从较大的培养瓶中进行子采样。该方案使用现成的材料来适应延长的培养时间，并允许在单个微孔板中接种多个重复。

an-adapted-optical-density-based-microplate-assay-for-characterizing

Research

JoVE Journal

Biology

An Adapted Optical Density-Based Microplate Assay for Characterizing Actinobacteriophage Infection

2.0K 次观看.  Ouachita Baptist University. 最终，我们对噬菌体 - 宿主相互作用感兴趣。该协议能够测量生长缓慢的细菌中的噬菌体感染，生成描述性的感染指标。该方法有助于研究生长缓慢的放线菌细菌中的噬菌体-宿主协同进化。快速微孔板方法对快速生长的细菌宿主提供了极好的结果，但太短，无法显示生长较慢的细菌宿主（如放线菌）中的细菌噬菌体感染进展。我们的方法适用于生长缓慢的细菌，允许细...

视频: 作者聚焦：一种用于表征放线菌噬菌体感染的基于光密度的微孔板检测  

Proper Care and Cleaning of the Microscope

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

适当的照顾和清洁的显微镜

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a ...

科研

生物学

Phase Contrast and Differential Interference Contrast (DIC) Microscopy

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by Zernike, in 1942, who received the Nobel prize for his achievement. DIC microscopy, introduced in the late 1960s, has been popular in biomedical research because it highlights edges of specimen structural detail, provides high-resolution optical sections of thick specimens including tissue cells, eggs, and embryos and does not suffer from the  phase halos  typical of phase-contrast images. This protocol highlights the principles and practical applications of these microscopy techniques.

Phase Contrast and Differential Interference Contrast DIC Microscopy

This protocol highlights the principles and practical applications of Phase and Differential Interference Contrast (DIC) Microscopy

相衬，微分干涉对比（DIC）显微镜

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by ...

Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example

Plants can resist herbivore damage through three broad mechanisms: antixenosis, antibiosis and tolerance1. Antixenosis is the degree to which the plant is avoided when the herbivore is able to select other plants2. Antibiosis is the degree to which the plant affects the fitness of the herbivore feeding on it1.Tolerance is the degree to which the plant can withstand or repair damage caused by the herbivore, without compromising the herbivore's growth and reproduction1. The durability of herbivore resistance in an agricultural setting depends to a great extent on the resistance mechanism favored during crop breeding efforts3.
We demonstrate a no-choice experiment designed to estimate the relative contributions of antibiosis and tolerance to spittlebug resistance in Brachiaria spp. Several species of African grasses of the genus Brachiaria are valuable forage and pasture plants in the Neotropics, but they can be severely challenged by several native species of spittlebugs (Hemiptera: Cercopidae)4.To assess their resistance to spittlebugs, plants are vegetatively-propagated by stem cuttings and allowed to grow for approximately one month, allowing the growth of superficial roots on which spittlebugs can feed. At that point, each test plant is individually challenged with six spittlebug eggs near hatching. Infestations are allowed to progress for one month before evaluating plant damage and insect survival. Scoring plant damage provides an estimate of tolerance while scoring insect survival provides an estimate of antibiosis. This protocol has facilitated our plant breeding objective to enhance spittlebug resistance in commercial brachiariagrases5.

Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example

This video explains mechanisms of host plant resistance to herbivory and demonstrates a no-choice test that estimates the relative contributions of antibiosis and tolerance to spittlebug resistance in Brachiaria spp.

表征草食动物的耐药机制:Spittlebugs臂形 SPP。为例

Plants can resist herbivore damage through three broad mechanisms: antixenosis, antibiosis and tolerance1. Antixenosis is the degree to which the plant is ...

An Explant Assay for Assessing Cellular Behavior of the Cranial Mesenchyme

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the neural tube in a process known as neurulation. During neurulation, morphogenesis of the mesenchyme that underlies the neural plate is believed to drive neural fold elevation. The cranial mesenchyme is comprised of the paraxial mesoderm and neural crest cells. The cells of the cranial mesenchyme form a pourous meshwork composed of stellate shaped cells and intermingling extracellular matrix (ECM) strands that support the neural folds. During neurulation, the cranial mesenchyme undergoes stereotypical rearrangements resulting in its expansion and these movements are believed to provide a driving force for neural fold elevation. However, the pathways and cellular behaviors that drive cranial mesenchyme morphogenesis remain poorly studied. Interactions between the ECM and the cells of the cranial mesenchyme underly these cell behaviors. Here we describe a simple ex vivo explant assay devised to characterize the behaviors of these cells. This assay is amendable to pharmacological manipulations to dissect the signaling pathways involved and live imaging analyses to further characterize the behavior of these cells. We present a representative experiment demonstrating the utility of this assay in characterizing the migratory properties of the cranial mesenchyme on a variety of ECM components.

The cranial mesenchyme undergoes dramatic morphogenic movements that likely provides a driving force for elevation of the neural folds1,2. Here we describe a simple ex vivo explant assay to characterize the cellular behaviors of the cranial mesenchyme during neurulation. This assay has numerous applications including being amenable to pharmacological manipulations and live imaging analyses.

为外植体含量的颅间充质细胞行为评定

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the ...

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

Marine elasmobranchs are valued animal models for biomedical and genomic studies as they are the most primitive vertebrates to have adaptive immunity and have unique mechanisms for osmoregulation 1-3. As the most primitive living jawed-vertebrates with paired appendages, elasmobranchs are an evolutionarily important model, especially for studies in evolution and development. Marine elasmobranchs have also been used to study aquatic toxicology and stress physiology in relationship to climate change 4. Thus, development and adaptation of methodologies is needed to facilitate and expand the use of these primitive vertebrates to multiple biological disciplines. Here I present the successful adaptation of RNA whole mount in situ hybridization and histological techniques to study gene expression and cell histology in elasmobranchs.
Monitoring gene expression is a hallmark tool of developmental biologists, and is widely used to investigate developmental processes 5. RNA whole mount in situ hybridization allows for the visualization and localization of specific gene transcripts in tissues of the developing embryo. The expression pattern of a gene's message can provide insight into what developmental processes and cell fate decisions a gene may control. By comparing the expression pattern of a gene at different developmental stages, insight can be gained into how the role of a gene changes during development.
While whole mount in situ's provides a means to localize gene expression to tissue, histological techniques allow for the identification of differentiated cell types and tissues. Histological stains have varied functions. General stains are used to highlight cell morphology, for example hematoxylin and eosin for general staining of nuclei and cytoplasm, respectively. Other stains can highlight specific cell types. For example, the alcian blue stain reported in this paper is a widely used cationic stain to identify mucosaccharides. Staining of the digestive tract with alcian blue can identify the distribution of goblet cells that produce mucosaccharides. Variations in mucosaccharide constituents on short peptides distinguish goblet cells by function within the digestive tract 6. By using RNA whole mount in situ's and histochemical methods concurrently, cell fate decisions can be linked to gene-specific expression.
Although RNA in situ's and histochemistry are widely used by researchers, their adaptation and use in marine elasmobranchs have met limited and varied success. Here I present protocols developed for elasmobranchs and used on a regular basis in my laboratory. Although further modification of the RNA in situ's hybridization method may be needed to adapt to different species, the protocols described here provide a strong starting point for researchers wanting to adapt the use of marine elasmobranchs to their scientific inquiries.

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

By combining methods for RNA whole mount in situ hybridization and histology, gene expression can be linked with cell fate decisions in the developing embryo. These methods have been adapted to marine elasmobranchs and facilitate the use of these animals as model organisms for biomedical, toxicology and comparative studies.

RNA<em在原位</em&gt;改编为海洋软骨鱼类杂交整装胚胎发育和细胞组织学

Marine  elasmobranchs are valued animal models for biomedical and genomic studies as  they are the most primitive vertebrates to have adaptive immunity ...

A Rapid and Specific Microplate Assay for the Determination of Intra- and Extracellular Ascorbate in Cultured Cells

Vitamin C (ascorbate) plays numerous important roles in cellular metabolism, many of which have only come to light in recent years. For instance, within the brain, ascorbate acts in a neuroprotective and neuromodulatory manner that involves ascorbate cycling between neurons and vicinal astrocytes - a relationship that appears to be crucial for brain ascorbate homeostasis. Additionally, emerging evidence strongly suggests that ascorbate has a greatly expanded role in regulating cellular and systemic iron metabolism than is classically recognized. The increasing recognition of the integral role of ascorbate in normal and deregulated cellular and organismal physiology demands a range of medium-throughput and high-sensitivity analytic techniques that can be executed without the need for highly expensive specialist equipment. Here we provide explicit instructions for a medium-throughput, specific and relatively inexpensive microplate assay for the determination of both intra- and extracellular ascorbate in cell culture.

Ascorbate plays numerous important roles in cellular metabolism, many of which have only come to light in recent years. Here we describe a medium-throughput, specific and inexpensive microplate assay for the determination of both intra- and extracellular ascorbate in cell culture.

快速，特异微孔板检测为内和细胞外抗坏血酸在培养细胞的测定

Vitamin C (ascorbate) plays numerous important roles in cellular metabolism, many of which have only come to light in recent years. For instance, within ...

Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera

Cell migration is an important process that influences many aspects of health, such as wound healing and cancer, and it is, therefore, crucial for developing methods to study the migration. The scratch assay has long been the most common in vitro method to test compounds with anti- and pro-migration properties because of its low cost and simple procedure. However, an often-reported problem of the assay is the accumulation of cells across the edge of the scratch. Furthermore, to obtain data from the assay, images of different exposures must be taken over a period of time at the exact same spot to compare the movements of the migration. Different analysis programs can be used to describe the scratch closure, but they are labor intensive, inaccurate, and forces cycles of temperature changes. In this study, we demonstrate an optimized method for testing the migration effect, e.g. with the naturally occurring proteins Human- and Bovine-Lactoferrin and their N-terminal peptide Lactoferricin on the epithelial cell line HaCaT. A crucial optimization is to wash and scratch in PBS, which eliminates the aforementioned accumulation of cells along the edge. This could be explained by the removal of cations, which have been shown to have an effect on keratinocyte cell-cell connection. To ensure true detection of migration, pre-treating with mitomycin C, a DNA synthesis inhibitor, was added to the protocol. Finally, we demonstrate the automated optical camera, which eliminates excessive temperature cycles, manual labor with scratch closure analysis, while improving on reproducibility and ensuring analysis of identical sections of the scratch over time.

Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera

Here we describe a procedure to test the cell migration in vitro with an automated optical camera microscope. The scratch assay has been widely used where the closure of the scratch is followed for a set period. The optical microscope enables dependable and cheap detection of cell migration.

用自动光学相机进行细胞迁移的体外试验优化划痕试验

Cell migration is an important process that influences many aspects of health, such as wound healing and cancer, and it is, therefore, crucial for ...

Single Cell Micro-aspiration as an Alternative Strategy to Fluorescence-activated Cell Sorting for Giant Virus Mixture Separation

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or fluorescence activated cell sorting (FACS) applied to the viral population. However, when the viruses in the mixture have similar morphologic properties and one of the viruses multiplies slowly, the presence of two viruses is discovered at the stage of genome assembly and the viruses cannot be separated for further characterization. To solve this problem, we developed a single cell micro-aspiration procedure that allows for separation and cloning of highly similar viruses. In the present work, we present how this alternative strategy allowed us to separate the small viral subpopulations of Clandestinovirus ST1 and Usurpativirus LCD7, giant viruses that grow slowly and do not lead to amoebal lysis compared to the lytic and fast-growing Faustovirus. Purity control was assessed by specific gene amplification and viruses were produced for further characterization.

Here, we describe a single cell micro-aspiration method for the separation of infected amoebae. In order to separate viral subpopulations in Vermamoeba vermiformis infected by Faustoviruses and unknown giant viruses, we developed the protocol detailed below and demonstrated its ability to separate two low-abundance novel giant viruses.

单细胞微吸气作为荧光激活细胞分类的替代策略，用于巨型病毒混合物分离

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or ...

An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis

Pathological choroidal angiogenesis, a salient feature of age-related macular degeneration, leads to vision impairment and blindness. Endothelial cell (EC) proliferation assays using human retinal microvascular endothelial cells (HRMECs) or isolated primary retinal ECs are widely used in vitro models to study retinal angiogenesis. However, isolating pure murine retinal endothelial cells is technically challenging and retinal ECs may have different proliferation responses than choroidal endothelial cells and different cell/cell interactions. A highly reproducible ex vivo choroidal sprouting assay as a model of choroidal microvascular proliferation was developed. This model includes the interaction between choroid vasculature (EC, macrophages, pericytes) and retinal pigment epithelium (RPE). Mouse RPE/choroid/scleral explants are isolated and incubated in growth-factor-reduced basal membrane extract (BME) (day 0). Medium is changed every other day and choroid sprouting is quantified at day 6. The images of individual choroid explant are taken with an inverted phase microscope and the sprouting area is quantified using a semi-automated macro plug-in to the ImageJ software developed in this lab. This reproducible ex vivo choroidal sprouting assay can be used to assess compounds for potential treatment and for microvascular disease research to assess pathways involved in choroidal micro vessel proliferation using wild type and genetically modified mouse tissue.

This protocol presents choroid sprouting assay, an ex vivo model of microvascular proliferation. This assay can be used to assess pathways involved in proliferating choroidal micro vessels and assess drug treatments using wild type and genetically modified mouse tissue.

眼部微血管血管生成前维沃胆球状芽分析

Pathological choroidal angiogenesis, a salient feature of age-related macular degeneration, leads to vision impairment and blindness. Endothelial cell ...

Imaging Molecular Adhesion in Cell Rolling by Adhesion Footprint Assay

Rolling adhesion, facilitated by selectin-mediated interactions, is a highly dynamic, passive motility in recruiting leukocytes to the site of inflammation. This phenomenon occurs in postcapillary venules, where blood flow pushes leukocytes in a rolling motion on the endothelial cells. Stable rolling requires a delicate balance between adhesion bond formation and their mechanically-driven dissociation, allowing the cell to remain attached to the surface while rolling in the direction of flow. Unlike other adhesion processes occurring in relatively static environments, rolling adhesion is highly dynamic as the rolling cells travel over thousands of microns at tens of microns per second. Consequently, conventional mechanobiology methods such as traction force microscopy are unsuitable for measuring the individual adhesion events and the associated molecular forces due to the short timescale and high sensitivity required. Here, we describe our latest implementation of the adhesion footprint assay to image the P-selectin: PSGL-1 interactions in rolling adhesion at the molecular level. This method utilizes irreversible DNA-based tension gauge tethers to produce a permanent history of molecular adhesion events in the form of fluorescence tracks. These tracks can be imaged in two ways: (1) stitching together thousands of diffraction-limited images to produce a large field of view, enabling the extraction of adhesion footprint of each rolling cell over thousands of microns in length, (2) performing DNA-PAINT to reconstruct super-resolution images of the fluorescence tracks within a small field of view. In this study, the adhesion footprint assay was used to study HL-60 cells rolling at different shear stresses. In doing so, we were able to image the spatial distribution of the P-selectin: PSGL-1 interaction and gain insight into their molecular forces through fluorescence intensity. Thus, this method provides the groundwork for the quantitative investigation of the various cell-surface interactions involved in rolling adhesion at the molecular level.

This protocol presents the experimental procedures to perform the adhesion footprint assay to image the adhesion events during fast cell rolling adhesion.