Name: deciphering the molecular mechanism and function of pore-forming toxins using leishmania major
Uploaded: 2022-10-28T14:00:00
Description: Watch this Scientific Journal Video about Deciphering the Molecular Mechanism and Function of Pore-Forming Toxins Using Leishmania major at JoVE.com

作者要感谢Keyel和Zhang实验室的成员对手稿的批判性审查。作者感谢艺术与科学学院显微镜学院使用设施。

所有适当的指南和标准的微生物学、安全性和细胞培养实践都用于使用和处理RG2病原体 利什曼原虫主要 和重组DNA。所有活的 L. major 实验均在BSL-2认证实验室的生物安全柜中进行。这项工作由德克萨斯理工大学机构生物安全委员会监督。
注意：从安全角度来看， 活的L. majorpromaputetes是风险组2病原体。使用机构生物安全委员会（IBC）的适当遏制，预防措施?...

<ol>
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本研究以人类病原虫大利 什曼原虫 为模型系统，描述了肺功能转移疗法分子机制和功能的研究方法。开发了一种基于中通量流式细胞术的细胞毒性测定法来测量单细胞活力。活力在人群水平上是定量的，因为LC50 值可以使用逻辑建模从剂量-反应曲线计算出来。作为原理验证，使用流式细胞术测定来说明培养基的选择可以改变野生型和鞘脂缺陷的 L.major 对SLO的敏感性，并确定...

孔形成毒素（PFTs）是细菌毒素中最大的家族1，但它们穿孔和破坏细胞的机制知之甚少。研究得最好的毛孔形成毒素家族是胆固醇依赖性溶细胞素（CDCs）。CDC主要由革兰氏阳性菌合成，包括坏死性筋膜炎的病原体化 脓性链球菌2。 化脓性 链球菌分泌CDC链球菌溶血素O（SLO），其作为单体与宿主细胞质膜中的甾醇结合，低聚化并将~20-30nm孔插入膜1。脂质在这一过程中所起的作用仍然不确定。
研究脂质-CDC相互作用的一种方法是使用化学定义的脂质体。虽然定义的脂质体提供了维持毒素结合和孔形成所需的脂质阈值的信息3，4，但它们并不能完全概括细胞功能。例如，重组脂质体缺乏哺乳动物宿主的脂质不对称性和响应毒素的脂质修饰5。脂质体的一种替代方案是使用哺乳动物细胞系。虽然这些细胞系在生理上更具相关性，但在毒素传感和抗性机制方面存在很大程度的冗余2。因此，用于抵抗CDC的修复途径仍然确定得很差。值得注意的是，Ca2+的流入是膜修复1的主要激活剂。在Ca2+涌入的下游，有多个途径参与，包括神经酰胺依赖性修复6，7和MEK依赖性修复途径6。这些途径与其他蛋白质效应物相互作用，包括运输所需的内体分选复合物（ESCRT）8和膜联蛋白6，9，10。由于冗余，在哺乳动物细胞中解剖这些途径具有挑战性，这混淆了数据解释。
在剖析修复途径的复杂性与简单性之间取得平衡的一种方法是使用更简单的生物，例如利什曼原虫属中的原生动物病原体。利什曼原虫在人类和其他动物中引起利什曼病。利什曼病的范围从皮肤利什曼病（自限性皮肤病变）到致命的内脏利什曼病（肝脾肿大），取决于物种和其他因素11。重型利什曼原虫是皮肤利什曼病的病原体，通过白蛉媒介传播给人类，用于了解利什曼原虫的功能和感染12。此外，利什曼原虫属是12。它们作为细胞内哺乳动物巨噬细胞寄生虫存在，称为无鞭毛虫，并在白蛉中作为自由游泳的鞭毛原鞭毛体存在12。主要前鞭毛乳杆菌可以在补充血清的培养基（如M199）中培养至高密度13。前乳鞭毛在遗传上也是可处理的;存在许多基因敲除，包括那些靶向脂质生物合成途径的基因敲除13。这些敲除可以通过感染Balb / c小鼠13来评估感染性和病变发展的生长和差异。
除了利什曼原虫培养的相对容易和脂质生物合成敲除的范围外，寄生虫的基因组比哺乳动物更简单。利什曼原虫最具特征的物种是L. major，它具有许多现有的遗传工具，例如脂质代谢缺陷的突变体14。值得注意的是，许多修复蛋白缺失。迄今为止，杆菌尚未发现关键哺乳动物修复蛋白（如膜联蛋白）的同系物。这使得能够表征进化上保守的修复途径，而没有哺乳动物系统的复杂性。然而，迄今为止，利什曼原虫尚未发现修复途径的特征。同时，参与修复的关键信号通路，如MEK通路6，在利什曼原虫sp.15，16中是保守的，尽管同系物需要验证。丝裂原活化蛋白激酶（MAPK）途径在墨西哥乳杆菌中得到了很好的研究，它有助于哺乳动物细胞的细胞内存活和热稳定性，并控制变环发生16。在利什曼原虫属中，15 种 MAPK 中有 10 种被表征为17.根据保守磷酸化唇序列中的身份，预测LmMAPK9和LmMAPK13与哺乳动物ERK1 / 2最相似。磷酸化唇序列对于哺乳动物ERK1 / 2和LmMAPK9和LmMAPK13都是TEY。然而，其中八个利什曼原虫MAPK具有TDY磷酸化基序15。在利什曼原虫、LmxMKK18 和 MEKK 相关激酶 （MRK1）19 中至少发现了两种 MEK 同系物。这表明在利什曼原虫中发现的见解可以转化为哺乳动物系统。在它们不能转化为哺乳动物系统的情况下，它们代表了治疗利什曼病的治疗靶点。
为了使用 L. majorpromasgotes 来研究膜修复和与毒素的相互作用，需要中等通量技术。虽然高分辨率活细胞成像能够实时可视化标记的蛋白质和膜，但它的通量很低，可能无法测量细胞存活率。中等通量活性测定包括通过流式细胞术测量染料摄取、线粒体活性测量或乳酸脱氢酶 （LDH） 等细胞蛋白的释放。在哺乳动物细胞中，LDH测定不能定量测量细胞死亡20。此外，基于群体的测定（如LDH释放或线粒体活性）不允许进行稳健的单细胞或多参数分析20。相比之下，基于流式细胞术的测定可实现多参数单细胞分析20。然而，这些测定尚未应用于了解毒素生物学或 对主要前鞭毛乳杆菌 毒素的反应。
在这项研究中，SLO被用作了解两种不同缓冲液中重 乳杆菌 鞘脂零突变体的质膜扰动的工具 - 通常用于培养 杆菌 的M199培养基和更简单的Tyrode缓冲液。描述了中等通量流式细胞术测定并用于生成毒素剂量-反应曲线。将流式细胞仪检测数据建模为逻辑曲线，以确定LC50 值。利用这些信息，可以确定SLO的亚溶解剂量，以便使用蛋白质印迹法验证MAPK抗体。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.2 mL microtiter (Marsh) tubes</td>
			<td>Fisher</td>
			<td>02-681-376</td>
			<td>Cytotoxicity assay</td>
		</tr>
		<tr>
			<td>1.5 mL microcentrifuge tube</td>
			<td>Fisher</td>
			<td>05-408-129</td>
			<td>Toxin dilutions</td>
		</tr>
		<tr>
			<td>15 mL centrifuge tube&#160;</td>
			<td>Avantor VWR (Radnor, PA)</td>
			<td>89039-666</td>
			<td>To hold cells and media</td>
		</tr>
		<tr>
			<td>1x Phosphate buffered saline (PBS)</td>
			<td>Fisher</td>
			<td>BP399</td>
			<td>For cell processing</td>
		</tr>
		<tr>
			<td>3% H2O2&#160;</td>
			<td>Walmart&#160; (Fayetteville, AR)</td>
			<td>N/A</td>
			<td>For ECL</td>
		</tr>
		<tr>
			<td>5x M199</td>
			<td>Cell-gro</td>
			<td>11150067</td>
			<td>Basal growth media for L. major promastigotes</td>
		</tr>
		<tr>
			<td>Biosafety cabinet</td>
			<td>Baker</td>
			<td></td>
			<td>To culture cells in sterile conditions</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Fisher</td>
			<td>BP1605-100</td>
			<td>Fraction V acceptable purity</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Fisher</td>
			<td>BP510-100</td>
			<td>Stock concentration 100 mM</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Thermo Fisher&#160;</td>
			<td>Heraeus Megafuge 40R</td>
			<td>To pellet the cells from culture</td>
		</tr>
		<tr>
			<td>Cy5 Mono-reactive dye pack</td>
			<td>Cytiva (Marlborough, MA)</td>
			<td>PA25031</td>
			<td>Fluorophore label for toxins</td>
		</tr>
		<tr>
			<td>Digital dry bath</td>
			<td>Benchmark</td>
			<td>BSH1002</td>
			<td>To denature protein samples</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Amresco</td>
			<td>0732-100G</td>
			<td>Stock concentration 0.5 M</td>
		</tr>
		<tr>
			<td>Excel</td>
			<td>Microsoft (Redmond, VA)</td>
			<td></td>
			<td>Data analysis software</td>
		</tr>
		<tr>
			<td>Flow cytometer (4-laser Attune NxT)</td>
			<td>Fisher</td>
			<td></td>
			<td>Cytometer for data acquisition</td>
		</tr>
		<tr>
			<td>FlowJo</td>
			<td>BD (Ashland, OR)</td>
			<td></td>
			<td>Software</td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Fisher</td>
			<td>BP531-500</td>
			<td>Fixative for counting cells</td>
		</tr>
		<tr>
			<td>G418</td>
			<td>Fisher</td>
			<td>BP673-1</td>
			<td>Selection agent for cells</td>
		</tr>
		<tr>
			<td>Hellmanex III</td>
			<td>Sigma</td>
			<td>Z805939</td>
			<td>Dilute 1:4 for cleaning cytometer</td>
		</tr>
		<tr>
			<td>Hemacytometer</td>
			<td>Fisher</td>
			<td>0267151B</td>
			<td>For counting cells</td>
		</tr>
		<tr>
			<td>Human red blood cells</td>
			<td>Zen-bio (Durham, NC)</td>
			<td>SER-10MLRBC</td>
			<td>To validate toxin activity</td>
		</tr>
		<tr>
			<td>Ice bucket</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Light microscope</td>
			<td>Nikon</td>
			<td>Eclipse 55i</td>
			<td>To visualize cells</td>
		</tr>
		<tr>
			<td>Nitrocellulose</td>
			<td>Fisher</td>
			<td>88018</td>
			<td>For probing proteins via antibodies</td>
		</tr>
		<tr>
			<td>Pipettors and tips</td>
			<td>Avantor VWR</td>
			<td></td>
			<td>To dispense reagents</td>
		</tr>
		<tr>
			<td>Power supply</td>
			<td>Bio-Rad</td>
			<td></td>
			<td>To run SDS-PAGE and transfers</td>
		</tr>
		<tr>
			<td>Propidium iodide</td>
			<td>Biotium</td>
			<td>40016</td>
			<td>Stock concentration 2 mg/mL in water</td>
		</tr>
		<tr>
			<td>Protein ladder</td>
			<td>Bio-Rad</td>
			<td>161-0373</td>
			<td>To determine molecular weight of proteins</td>
		</tr>
		<tr>
			<td>SDS-PAGE Running Apparatus (Mini Protean III)</td>
			<td>Bio-Rad</td>
			<td>165-3302</td>
			<td>To separate proteins based on their size</td>
		</tr>
		<tr>
			<td>Sealing tape</td>
			<td>R&#38;D</td>
			<td>DY992</td>
			<td>To seal plates with cells</td>
		</tr>
		<tr>
			<td>Streptolysin O C530A plasmid insert</td>
			<td></td>
			<td></td>
			<td>Cloned into pBAD-gIII vector (Reference: 7)</td>
		</tr>
		<tr>
			<td>Streptolysin O C530A toxin</td>
			<td>Lab purified</td>
			<td></td>
			<td>Specific activity&#160; 4.34 x 105 HU/mg</td>
		</tr>
		<tr>
			<td>Swinging bucket rotor</td>
			<td>Thermo Fisher&#160;</td>
			<td>75003607</td>
			<td>To centrifuge cells</td>
		</tr>
		<tr>
			<td>V-bottom plate</td>
			<td>Greiner Bio-one</td>
			<td>651206</td>
			<td>For cytotoxicity assay</td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>Benchmark</td>
			<td>BV1000</td>
			<td>To mix cells</td>
		</tr>
		<tr>
			<td>Western blot imaging system (Chemi-doc)</td>
			<td>Bio-Rad</td>
			<td></td>
			<td>To visualize proteins by western blot</td>
		</tr>
		<tr>
			<td>Western Blot Transfer Apparatus (Mini Protean III)</td>
			<td>Bio-Rad</td>
			<td>170-3930</td>
			<td>Transfer proteins to nitrocellulose</td>
		</tr>
		<tr>
			<td>Whatman Filter paper</td>
			<td>GE Healthcare Life Sciences</td>
			<td>3030-700</td>
			<td>Used in transfer of proteins to nitrocellulose</td>
		</tr>
		<tr>
			<td>Antibody</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-ERK antibody</td>
			<td>Cell Signaling Technologies&#160;</td>
			<td>Cat# 9102S</td>
			<td>Rabbit (1:1000 dilution)</td>
		</tr>
		<tr>
			<td>Anti-lipophosphoglycan (LPG) antibody</td>
			<td>CreativeBioLabs&#160;</td>
			<td>Cat# WIC79.3</td>
			<td>Mouse (1: 1000)</td>
		</tr>
		<tr>
			<td>Anti-MEK antibody</td>
			<td>Cell Signaling Technologies&#160;</td>
			<td>Cat# 9122L</td>
			<td>Rabbit (1:1000)</td>
		</tr>
		<tr>
			<td>Anti-mouse IgG, HRP conjugate</td>
			<td>Jackson Immunoresearch&#160;</td>
			<td>Cat#715-035-151</td>
			<td>Donkey (1:10000)</td>
		</tr>
		<tr>
			<td>Anti-phosphoERK antibody</td>
			<td>Cell Signaling Technologies&#160;</td>
			<td>Cat# 9101S</td>
			<td>Rabbit (1:1000)</td>
		</tr>
		<tr>
			<td>Anti-pMEK antibody</td>
			<td>Cell Signaling Technologies&#160;</td>
			<td>Cat# 9121S</td>
			<td>Rabbit (1:1000)</td>
		</tr>
		<tr>
			<td>Anti-rabbit IgG, HRP conjugate</td>
			<td>Jackson Immunoresearch&#160;</td>
			<td>Cat#711-035-152</td>
			<td>Donkey (1:10000)</td>
		</tr>
		<tr>
			<td>Anti-tubulin antibody</td>
			<td>Sigma</td>
			<td>Cat# T5168</td>
			<td>Mouse (1: 2000)</td>
		</tr>
		<tr>
			<td>Leishmania major Genotypes&#160;</td>
			<td></td>
			<td></td>
			<td>Reference: 13</td>
		</tr>
		<tr>
			<td>Episomal addback (spt2-/+SPT2)</td>
			<td></td>
			<td></td>
			<td>&#916;spt2::HYG/&#916;spt2:PAC/+pXG-SPT2</td>
		</tr>
		<tr>
			<td>Serine palmitoyltransferase subunit 2 knockout (spt2-)</td>
			<td></td>
			<td></td>
			<td>&#916;spt2::HYG/&#916;spt2::PAC&#160;</td>
		</tr>
		<tr>
			<td>Wild type (WT)</td>
			<td></td>
			<td></td>
			<td>LV39 clone 5 (Rho/SU/59/P)</td>
		</tr>
	</tbody>
</table>

deciphering the molecular mechanism and function of pore-forming toxins using leishmania major

了解造孔毒素（PFT）的功能和机制具有挑战性，因为细胞可以抵抗PFT引起的膜损伤。虽然生物物理方法有助于理解孔的形成，但它们通常依赖于缺乏膜脂质和蛋白质全部补充的还原论方法。培养的人类细胞提供了一种替代系统，但它们的复杂性和修复机制的冗余使得识别特定机制变得困难。相比之下，导致皮肤利什曼病的人类原生动物病原体，即大利什曼原虫，在复杂性和生理相关性之间提供了最佳平衡。杆菌在遗传上是可处理的，可以在体外培养到高密度，并且可以在已建立的小鼠模型中测量扰动对感染的任何影响。此外，L. major合成的脂质与哺乳动物不同，这可能会改变膜动力学。膜动力学的这些改变可以用来自最佳特征毒素家族胆固醇依赖性溶细胞素（CDC）的PFT进行探测。CDC与利什曼原虫膜中的麦角甾醇结合，可杀死大鞭毛乳杆菌，表明杆菌是确定PFT功能的细胞和分子机制的合适模型系统。这项工作描述了在L. majorpromagotes中测试PFT功能的方法，包括寄生虫培养，用于评估脂质敏感性的遗传工具，膜结合测定和细胞死亡测定。这些测定将能够快速使用L. major作为强大的模型系统，用于了解一系列进化上多样化的生物体的PFT功能和脂质组织的共性。

与M199相比，Tyrode缓冲液中促乳腺对SLO的敏感性增加 比较不同测定缓冲液之间主要鞭毛乳杆菌的SLO敏感性。在流式细胞仪上分析之前，在无血清M199或补充有2mM CaCl2的Tyrode缓冲液中用SLO攻击野生型，spt2-和spt2-/+ SPT2前鞭毛体30分钟。适合分析的寄生虫是通过前向/侧面散射图鉴定的单细胞（图3A，B）。使用?...

Watch this Scientific Journal Video about Deciphering the Molecular Mechanism and Function of Pore-Forming Toxins Using Leishmania major at JoVE.com

Deciphering the Molecular Mechanism and Function of Pore-Forming Toxins Using Leishmania major

这里介绍的是使用 利什曼原虫主要 前鞭毛体来确定孔形成毒素诱导的结合、细胞毒性和信号传导的方案。提供了链球菌溶血素O的概念验证。其他毒素也可用于利用 杆菌 中可用的遗传突变体来确定毒素抗性的新机制。

利用利什曼原虫破译毛孔形成毒素的分子机制和功能

Understanding the function and mechanism of pore-forming toxins (PFTs) is challenging because cells resist the membrane damage caused by PFTs. While ...

对毛孔形成毒素的抵抗机制知之甚少。我们的协议能够使用大利什曼原虫（一种遗传上易于处理和生理相关的系统）来形成毒素的功能和机制。细胞毒性测定的主要优点是在实时受到毒素攻击时，在中通量测定中比较单细胞水平上多种表型的细胞活力。两性霉素 B 等膜扰动剂是利什曼原虫的一线疗法。该测定能够鉴定增强两性霉素B和/或新的膜损伤剂的药物。该测定提供了对修复反应和与修复反应相关的信号通路等领域的见解。该测定可应用于原生动物，如锥虫和福氏内格勒菌。人们发现很难理解毒素系列稀释系列。因此，我能给出的最佳建议是在设置实验之前绘制系列稀释系列的示意图。首先，将0.5毫升处理过的鞭毛体保留在单独的管中作为未染色的对照。向剩余的前鞭毛体中加入每毫升2毫克碘化丙啶或PI，最终浓度为每毫升10微克。涡旋三秒钟。将处理好的鞭毛加入V底，96孔板或沼泽管的每个孔中，并将板或管架以大约45度角放置在冰上。向每个无毒素对照中加入100微升碘化丙啶测定缓冲液。通过直观地识别总体积为 200 微升且颜色较深的管，验证是否正确添加了对照。将毒素体积添加到最高稀释度。然后，连续稀释毒素。上下移液至少八次以确保混合。从最低毒素浓度开始，快速将100微升毒素添加到正确的行中，并继续直到所有毒素都添加到细胞中。用密封胶带密封板。在37摄氏度下孵育30分钟后，包装并将板运输到流式细胞仪。对于数据采集，首先根据制造商的说明和设施政策设置流式细胞仪和采集软件。使用未染色的L.major前鞭毛样品，根据所选染料设置前向和侧向散射的门以及初始荧光参数。使用单染色对照，设置活性染料和任何荧光标记毒素的闸门。监测微堵塞的前向散射与时间的关系，并在细胞仪上为每个样品采集超过10， 000个门控事件。对于数据分析，根据需要通过对前向和侧向散射和时间进行门控来控制总单细胞L.主要前鞭毛体。按照流式细胞仪的建议使用高度或面积。识别死细胞并将其选为PI高。在Excel中组织剂量 - 反应曲线进行分析。确定两个技术重复之间每个条件的平均PI高百分比。包括毒素浓度和平均百分比特异性裂解，以及实验细节和/或原始百分比PI高计算。将另外四列标记为建模、残差、参数和参数值。验证第一列是否与实验参数、毒素浓度和比裂解百分比相对应。通过在“参数值”列中输入值来初始化参数 L、k 和 c。在“建模”列中，创建逻辑模型。在残差列中，计算建模数值与实际特定裂解之间的差值的平方。在参数值列的 SUM 旁边，对残差列中的所有值求和。在“参数值”列中，在LC50旁边，初始化公式以根据确定的值计算LC50。从数据选项卡中打开规划求解。选择设置目标作为包含所计算残差总和的像元。将其设置为最小值。更改参数值 L、k 和 c 的变量单元格。对于负 k 值，通过从 k 中分解出负值来修改方程，以将 k 更改为正。使用 GRG 非线性求解方法，然后单击求解。检查曲线，并自动计算LC50。通过图形绘制百分比特异性裂解和针对毒素浓度的建模来验证拟合。鞘脂缺陷的SPT2敲除前鞭毛对无血清M199和Tyrode缓冲液中的SLO敏感。野生型和SPT2回加原鞭毛在无血清M199中对SLO具有抗性，但在Tyrode缓冲液中的特异性裂解率低于20%。SPT2敲除前鞭毛对Tyrode缓冲液中SLO的敏感性大约是M199的八倍。这些数据表明，毒素敏感性可能因使用的缓冲液而异。在L.major前鞭毛体中观察到磷酸化MEK的120千道尔顿带。对于总MEK，观察到约120千道尔顿和55千道尔顿的条带，分别与MRK1和LmxMKK的大小一致。磷酸化-ERK检测到相似的条带，而ERK抗体染色在该测定中并不可靠。尝试此过程时要记住的最重要的事情是确保正确处理毒素。利什曼原虫可用作研究质膜修复的遗传学模型，为白蛉中肠利什曼原虫细菌相互作用期间的膜修复机制提供新的见解。

Research

JoVE Journal

Immunology and Infection

Deciphering the Molecular Mechanism and Function of Pore-Forming Toxins Using Leishmania major

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

在Live主机转利什曼原虫寄生虫在体内成像

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most ...

科研

免疫与感染

Determination of Molecular Structures of HIV Envelope Glycoproteins using Cryo-Electron Tomography and Automated Sub-tomogram Averaging

使用低温电子断层扫描和自动子断层扫描平均HIV包膜糖蛋白分子结构的测定

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) (www.usaid.gov). The ...

Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay

枚举主要外周血白细胞人口多中心临床试验，使用全血表型分析

Cryopreservation of peripheral blood leukocytes is widely used to preserve cells for immune response evaluations in clinical trials and offers many ...

In Situ Detection of Autoreactive CD4 T Cells in Brain and Heart Using Major Histocompatibility Complex Class II Dextramers

In Situ Detection of Autoreactive CD4 T Cells in Brain and Heart Using Major Histocompatibility Complex Class II Dextramers

原位检测自身反应性CD4 + T细胞在大脑和心脏使用主要组织相容性复合II类Dextramers的

This report demonstrates the use of major histocompatibility complex (MHC) class II dextramers for detection of autoreactive CD4 T cells in situ in myelin ...

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

肝脏活体如下显微成像<em&gt;利什曼原虫</em&gt;感染：肝脏血流动力学的评估

Intravital microscopy (IVM) is a powerful optical imaging technique that has made possible the visualization, monitoring and quantification of various ...

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

破译和成像发病机制与盘带<em&gt;脓肿分枝杆菌</em&gt;斑马鱼胚胎

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen ...

Implementation of a Permeable Membrane Insert-based Infection System to Study the Effects of Secreted Bacterial Toxins on Mammalian Host Cells

基于插入渗透膜感染系统的实现研究分泌细菌病毒对哺乳动物宿主细胞的作用

Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate ...

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

评估视网膜小胶质细胞吞噬功能<em&gt;在体内</em&gt;使用基于流式细胞术流量测定

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

Unravelling the Function of a Bacterial Effector from a Non-cultivable Plant Pathogen Using a Yeast Two-hybrid Screen

从非耕种植物病原菌利用酵母双杂交筛选Unravelling一个细菌效应中的作用

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

在体内应用免疫模型检测抗原特异性 T 细胞细胞功能的试验

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...