作者希望感谢何塞·路易斯·拉莫斯-巴尔德拉斯的技术支持。这项工作得到了CONACYT（FORDECYT-PRONACE-303027）对PLL的以下资助的支持。OM-P和EO-M获得了CONACYT的研究金（分别为736162和481437）。MCM-M 获得了 CONACYT 的奖学金（墨西哥 2022 年 （3））。

这里描述的所有动物实验都得到了墨西哥国立自治大学细胞生理学研究所动物处理内部委员会（CICUAL）的批准。
注意:整个协议的流程图如图 1 所示。
1. 老鼠的住房
<ol><li>使用8-10周龄的雌性或雄性小鼠组，在12小时光照/黑暗循环中饲养在具有恒定温度和湿度的动物设施中， 随意获取水和 食物。 注意:该协议在C57BL / 6背景中使用IL-9报告小鼠品系，如前所述4;然而，可以使用其他IL-9报告菌株6，7，8，以及细胞内IL-9染色，结果各不相同9。</li>
</ol>2.小鼠感染
<ol><li>在感染前1天将小鼠的背部从身体中间剃到尾巴底部附近。麻醉剂的使用不是必需的。</li>
	<li>如前所述2，11，在下背部的100μL磷酸盐缓冲盐水（PBS）10中皮下接种每只小鼠200个活的第三阶段巴西猪笼草幼虫（L3），或单独注射100μLPBS作为对照。在感染后第 4 天、第 7 天或第 10 天处死动物。</li>
</ol>3.肺、小肠、纵隔、肠系膜淋巴结分离
<ol><li>通过颈椎脱位对小鼠实施安乐死。将鼠标放在背上，喷洒70%乙醇。用剪刀做一个中线切口，打开皮肤，露出腹部和胸部区域。 注意:异氟醚可用作替代安乐死方法12。不建议使用二氧化碳室，因为 CO2 会导致肺组织损伤和出血13。</li>
	<li>分离纵隔淋巴结和肺<ol><li>在胸骨处切开一个切口，切成"V"形，以去除肋骨和胸肌。一旦胸腔暴露，将纵隔淋巴结定位在食道旁边，心脏下方（见 补充图1）。</li>
		<li>提取纵隔淋巴结并将其收集在12孔培养板中的1mL R-10培养基（表1）中。在冰上保存，避光直至加工。 注意:样品应避光，以避免减少这些实验中使用的报告小鼠的荧光信号。</li>
		<li>提取肺并将其收集在每只小鼠12孔培养板中的1mL R-10培养基中。在冰上保存，避光直至加工。</li>
	</ol></li>
	<li>分离肠系膜淋巴结链和小肠<ol><li>暴露腹膜腔，并小心地将小肠向右移动，以暴露沿结肠的肠系膜淋巴结（MLN）链。</li>
		<li>用镊子取出MLN，在纸巾上轻轻滚动，然后拉下脂肪。</li>
		<li>将 MLN 转移到 12 孔培养板中的 1 mL R-10 培养基中。在冰上保存，避光直至加工。</li>
		<li>切开幽门括约肌下方和盲肠上方的小肠。在镊子的帮助下慢慢拉出肠道，去除附着的肠系膜和脂肪组织。</li>
		<li>将小肠放在纸巾上，并用PBS大量润湿。用剪刀从小肠上取下佩耶氏贴片。 注意:为了保持活力，请去除剩余的脂肪组织，并在整个过程中用PBS保持小肠湿润。</li>
		<li>用剪刀纵向切开小肠，将镊子轻轻滑过开放的肠，去除粪便和粘液。</li>
		<li>用镊子握住小肠，小心地将其浸入冰上的 5 mL PBS 中洗涤几次。重复两次。</li>
		<li>将小肠切成短块（约5毫米），并将它们收集在50 mL锥形管中，其中含有10 mL HBSS14 和2%FBS（表1）。立即继续分离上皮内和固有层细胞。</li>
	</ol></li>
</ol>4.制备小肠、肺和淋巴结的单细胞悬液
注意:从小肠制备单细胞悬浮液时，每人最多处理六只小鼠非常重要，因为细胞活力随着处理时间的延长而显着降低。该方法改编自 Heligmosomoides polygyrus 感染小鼠模型5。
<ol><li>在37°C下预热振荡培养箱，R-20培养基，HBSS和HBSS-2mM EDTA（表1）。</li>
	<li>每个样品制备10 mL小肠消化培养基（表1）。</li>
	<li>用手用力摇晃步骤3.3.8中的肠片。</li>
	<li>通过玻璃漏斗上的尼龙网（约10 cm x 10 cm）过滤每个样品。通过在网眼上加入 10 mL 预热的 HBSS 来洗涤样品并丢弃流通物。再重复一次。 注意:为每个样品使用一个新的网格，并在整个过程中重复使用它。</li>
	<li>从漏斗中取出网布，并将网孔中的样品收集在装有10 mL温热HBSS-2 mM EDTA的50 mL锥形管中（步骤4.1）。在37°C孵育10分钟，以200rpm振荡。</li>
	<li>以最大速度 （3，200 rpm） 涡旋 10 秒，并在玻璃漏斗上用网状物过滤样品，回收 50 mL 锥形管中的流出物。</li>
	<li>重复步骤 4.5 和 4.6 两次，在同一 50 mL 锥形管中回收流出物。上皮内细胞位于这个 30 mL 级分中。保存剩余的组织。</li>
	<li>上皮内细胞的单细胞悬液制备<ol><li>在室温（RT）下以450× g 离心在步骤4.7中回收的30mL细胞悬液5分钟。弃去上清液。</li>
		<li>加入 5 mL PBS 并在室温下以 450 x g 离心 5 分钟。 弃去上清液。</li>
		<li>将细胞沉淀重悬于3 mL RPMI 10%FBS-20 μg/mL DNase中（表1）。保持在冰上，避光直到细胞染色。</li>
	</ol></li>
	<li>由固有细胞层制备单细胞悬液<ol><li>通过将超过 10 mL 的温热 HBSS 倒入漏斗上的网状物来洗涤步骤 4.7 中剩余的小肠组织。重复洗涤。将网状物中的组织收集在装有 10 mL 小肠消化培养基的 50 mL 锥形管中。</li>
		<li>在37°C孵育30分钟，以200rpm振荡。每5分钟以最大速度涡旋10秒。</li>
		<li>加入 10 mL FACS-EDTA 缓冲液（表 1）以停止消化反应，并将其置于冰上。</li>
		<li>使用血清移液管通过 100 μm 细胞过滤器过滤每个样品，在放置在冰上的 50 mL 锥形管中回收悬浮液。</li>
		<li>在4°C下以600× g 离心6分钟。</li>
		<li>弃去上清液。用5mL PBS洗涤细胞沉淀，并在4°C下以600× g 离心6分钟。</li>
		<li>弃去上清液，并将细胞沉淀重悬于 1 mL RPMI 10% FBS-20 μg/mL 脱氧核糖核酸酶中。保持在冰上，避光直到细胞染色。</li>
	</ol></li>
	<li>肺单细胞悬液制备 注意:每个肺和小肠应由两个人并行处理，以避免延长处理时间，从而导致细胞活力低下。<ol><li>每个处理的样品准备 4 mL 肺消化培养基（表 1）。</li>
		<li>从步骤3.2.3中从含有肺的孔中取出RPMI培养基。用细剪刀将肺切成小块。</li>
		<li>用刮刀将肺碎片转移到 15 mL 锥形管中。加入4 mL肺消化培养基（表1）。</li>
		<li>在37°C下孵育30分钟，以250rpm振荡。完成后，继续在冰上，直到每个样品都得到处理。</li>
		<li>通过放置在6孔培养板的单孔中的100μm细胞过滤器过滤每个样品。用注射器柱塞解离组织。</li>
		<li>在 15 mL 锥形管中回收每个样品，并加入 4 mL R-2 培养基（表 1）。在处理其他样品时保持冰上。</li>
		<li>在4°C下以600× g 离心5分钟。 弃去上清液并将细胞沉淀重悬于1mL R-5培养基中（表1）。</li>
		<li>离心时，每个样品制备4 mL的27.5%密度梯度溶液（表1），以富集造血细胞级分15，16。与其他类似的密度梯度培养基相比，这种单细胞分离策略更有效、更具成本效益且毒性更小17.</li>
		<li>将4mL的27.5%密度梯度溶液加入步骤4.10.7的1mL细胞悬液中，并剧烈摇晃。</li>
		<li>在混合悬浮液上缓慢加入 1 mL R-5 培养基（表 1）以形成两相。</li>
		<li>在室温下以 1，500 x g 离心 20 分钟，低加速度和制动器关闭。观察两相之间形成的环。</li>
		<li>用 1 mL 微量移液器回收两相之间形成的环，并重悬于 4 mL R-2 培养基中。</li>
		<li>在4°C下以450× g 离心5分钟。 弃去上清液。将细胞沉淀重悬于1mL ACK缓冲液中（表1），并在室温下孵育1分钟。</li>
		<li>加入4mL的R-5培养基，并在4°C下以450× g 离心5分钟。 弃去上清液并将细胞沉淀重悬于1mL R-10培养基中。保存在冰上，避光直至染色以进行流式细胞术。</li>
	</ol></li>
	<li>淋巴结单细胞悬液制备<ol><li>将步骤3.2.2或3.3.3中的淋巴结放在6孔培养板中的两片网片之间，并用注射器柱塞解离。</li>
		<li>在1.5mL锥形管中回收细胞悬液，并在4°C下以450× g 离心5分钟。</li>
		<li>弃去上清液，并将细胞沉淀重悬于1mL R-10培养基中。保存在冰上，避光直至染色以进行流式细胞术。 注意:如果可见团块，请通过100μm细胞过滤器过滤样品。</li>
	</ol></li>
</ol>5.流式细胞术的细胞染色（图2和图3）
注意:将步骤4.11.3中的淋巴结细胞悬液在450× g 下在4°C下离心5分钟，并将细胞沉淀重悬于500μLFACS缓冲液中（表1）。
<ol><li>用于鉴定ILC2的细胞染色（图3 和 补充图2） 注意:此染色程序特定于鉴定表达IL-9的ILC2细胞。<ol><li>在96孔锥形底部培养板中，从步骤4.10.14中板每个肺样品（约1.8 x 10 6个细胞）板150μL，从步骤4.11.3中板每个淋巴结样品50μL（纵隔和肠系膜淋巴结样品分别约为0.7 x 10 6和2.2 x 106细胞）。加入 100 μL FACS 缓冲液，并在 4 °C 下以 450 x g 离心 5 分钟。</li>
		<li>在96孔锥形底部培养板中，来自步骤4.8.3和4.9.7的每个小肠样品板100μL（上皮内和固有层样品分别约为2.7 x 106和 0.6 x 106 细胞）。加入 150 μL FACS 缓冲液，并在 4 °C 下以 450 x g 离心 5 分钟。</li>
		<li>弃去上清液，并将每个细胞沉淀重悬于在FACS缓冲液中稀释的50μL生物素化抗体混合物（表2）中。在4°C避光孵育30分钟。 注意:使用含有20μg/ mL DNase的FACS缓冲液用于上皮内和固有层样品。</li>
		<li>加入 150 μL FACS 缓冲液。在4°C下以450× g 离心5分钟。</li>
		<li>弃去上清液，并将细胞沉淀重悬于 200 μL FACS 缓冲液中。再次离心并弃去上清液。</li>
		<li>将细胞沉淀重悬于50μL抗体/染色混合物中（表2），并在4°C避光下孵育30分钟。</li>
		<li>加入 150 μL FACS 缓冲液。在4°C下以450× g 离心5分钟。</li>
		<li>弃去上清液，并将细胞沉淀重悬于 200 μL FACS 缓冲液中。再次离心并弃去上清液。重复洗涤（步骤5.1.7），弃去上清液。</li>
		<li>将细胞沉淀重悬于 300 μL FACS 缓冲液中，并通过流式细胞术进行分析。</li>
		<li>对于小肠和肺样本，使用门控策略:淋巴细胞，单细胞，活细胞，造血细胞，CD90 +谱系细胞，ST2 +细胞和IL-9 +细胞（图3A，B 和 补充图2A）。对于淋巴结样本，使用门控策略:活细胞，单细胞，CD90 +谱系细胞，ST2 +细胞和IL-9 +细胞（补充图2B，C）。</li>
	</ol></li>
	<li>用于鉴定产生IL-9的淋巴细胞的细胞染色（图2 和 补充图3）<ol><li>将步骤 4.10.14 的每个肺样品转移 800 μL 到 1.5 mL 管（约 14.6 x 106 个细胞）中。在4°C下以450× g 离心5分钟。 弃去上清液。</li>
		<li>对于淋巴结样品，将步骤 4.11.3 中的 400 μL 细胞悬液（纵隔和肠系膜淋巴结样品分别为约 5.6 x 10 6 和 17.5 x 106 细胞）接种在 96 孔锥形底板中，分两步进行。<ol><li>首先转移200μL，在4°C下以450× g 离心5分钟，弃去上清液。</li>
			<li>将另外 200 μL 转移到相应的孔中。在4°C下以450× g 离心5分钟，弃去上清液。</li>
		</ol></li>
		<li>将细胞沉淀重悬于50-400μL抗体/染色混合物中（表3），并在4°C避光下孵育30分钟。 注意:肺样本应重悬于400μL中，纵隔淋巴结样本重悬于50μL中，肠系膜淋巴结样本重悬于100μL染色混合物中。</li>
		<li>向纵隔淋巴结样品中加入 150 μL FACS 缓冲液，向肠系膜淋巴结样品中加入 100 μL 的 FACS 缓冲液。在4°C下以450× g 离心5分钟，弃去上清液。</li>
		<li>通过将沉淀分别重悬于 1 mL 和 200 μL FACS 缓冲液中来洗涤肺和淋巴结样品。在4°C下以450×g 离心5分钟。 弃去上清液并重复洗涤。</li>
		<li>将肺样品重悬于 600 μL FACS 缓冲液中，并通过流式细胞术进行分析。使用门控策略:淋巴细胞，单细胞，活细胞，造血细胞，CD4 + TCRβ +细胞和IL-9 +细胞（图2A）。</li>
		<li>将淋巴结样品重悬于 300 μL FACS 缓冲液中，并通过流式细胞术进行分析。使用门控策略:淋巴细胞，单细胞，活细胞，CD4 + TCRβ +细胞和IL-9 +细胞（图2B 和 补充图3A）。</li>
	</ol></li>
</ol>6. 单细胞悬浮液中细胞绝对数量的测定
<ol><li>用 PBS 以 1:20 的比例（10 μL 样品 + 190 μL PBS）稀释分离步骤 4.8.3、4.9.7、4.10.14 和 4.11.3 中的样品。</li>
	<li>将步骤 6.1 中的每个稀释样品 10 μL 与 10 μL 台盼蓝混合。将 10 μL 加载到血细胞计数器中并计数活细胞，考虑每次稀释。</li>
	<li>通过将流式细胞术确定的活细胞（活力染料阴性细胞）中感兴趣的群体百分比乘以分离后单细胞悬液中的活细胞总数，然后将该数字除以100（补充图4，补充图5和补充图6），获得细胞的绝对数。 绝对数=（通过流式细胞术测定的活细胞目标群体的百分比x单细胞悬液中的活细胞总数）/100</li>
</ol>

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作者没有什么可透露的。

要全面了解肠道寄生虫-宿主相互作用和对蠕虫感染的免疫反应，需要鉴定和分析不同的细胞群和效应分子，这些细胞群和效应分子是诱导组织重塑和蠕虫排出的关键。土壤传播的蠕虫感染是全世界发展中国家的一个大问题。然而，直到最近，还没有允许分析受这种感染影响的主要器官小肠中存在的稀有细胞群的方案5。该协议涵盖了先前描述的方法，这些方法允许在 巴西奈瑟...

钩虫是肠道寄生虫，感染全世界约7亿人，主要在不发达国家的热带地区。 十二指肠钩瘤 和美洲钩虫（人类最常见的 钩虫寄生虫）的高强度感染会导致贫血和蛋白质缺乏，从而导致生长和智力发育延迟1。 美洲猪笼草 和啮齿动物寄生虫巴西 圆线虫 在其宿主中诱导原型2型免疫反应，并且在其生命周期中具有相似性。因此， 巴西猪笼草 小鼠感染是人类钩虫感染最常用的模型。3期（L3） 巴西猪笼草 感染性幼虫在感染后的最初几个小时内从皮肤移动到肺部。一旦进入肺部，它们就会变成L4并沿着气管向上迁移，被吞咽，通过胃，并在4-5天内到达肠道成为成虫（L5）。在肠道中，L5蠕虫产卵，这些卵在粪便中排泄以重新启动寄生虫生命周期2。
巴西猪笼草诱导的免疫应答的特征是几种 2 型细胞因子增加，包括 IL-4、IL-5、IL-9、IL-10 和 IL-13，以及嗜酸性粒细胞增多、嗜碱性粒细胞增多、杯状细胞和肥大细胞增生，以及 IgG1 和 IgE 生成增加。大多数试图识别和定义巴西奈瑟菌感染时引发的免疫反应的研究都集中在IL-4或IL-13在该模型3中的作用上。然而，表达IL-9的细胞的鉴定和表征以及这种细胞因子的功能在很大程度上被忽视了，直到Licona-Limón等人发表了第一项研究，证明IL-9在针对巴西猪笼草的免疫反应中的关键作用。本研究使用报告小鼠将T细胞（主要是辅助性T细胞9）和2型先天淋巴细胞（ILC2）描述为感染时表达IL-9的主要细胞亚群4。
从蠕虫感染的肺部分离和表征免疫细胞是可行的，并且已被广泛报道3，4。然而，由于固有的组织重塑和粘液的产生，在受感染的肠道中这样做被证明是一个技术挑战，直到最近Ferrer-Font等人5的发表。该小组概述了一种方案，用于分离和分析来自 Heligmosomoides polygyrus感染的鼠肠的免疫亚群的单细胞悬浮液。基于此，我们现在已经标准化了从 巴西猪笼草 感染肠道中分离和细胞术分析表达IL-9的淋巴细胞的方案。此外，我们已经建立了来自整个感染过程中不同细胞来源和解剖位置的IL-9动力学。
表征参与这种感染的不同细胞群对于更广泛地了解对寄生虫的免疫反应及其与宿主的相互作用至关重要。这个全面的方案提供了一条明确的途径，可以在感兴趣的疾病阶段从所需器官中分离和分析产生IL-9的细胞，从而可以显着提高有关这些细胞在 巴西猪笼草 感染和寄生虫感染中的作用的知识。

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			<td>ACK buffer</td>
			<td>Homemade</td>
			<td></td>
			<td></td>
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		<tr>
			<td>Attune Nxt cytometer</td>
			<td>Thermofisher</td>
			<td></td>
			<td></td>
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		<tr>
			<td>B220</td>
			<td>Biolegend</td>
			<td>103204</td>
			<td></td>
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		<tr>
			<td>CD11b</td>
			<td>Biolegend</td>
			<td>101204</td>
			<td></td>
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		<tr>
			<td>CD11c&#160;</td>
			<td>Biolegend</td>
			<td>117304</td>
			<td></td>
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			<td>CD19&#160;</td>
			<td>Biolegend</td>
			<td>115504</td>
			<td></td>
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			<td>CD4</td>
			<td>Biolegend</td>
			<td>100404</td>
			<td></td>
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		<tr>
			<td>CD4 (BV421)</td>
			<td>Biolegend</td>
			<td>100443</td>
			<td></td>
		</tr>
		<tr>
			<td>CD45.2</td>
			<td>Biolegend</td>
			<td>109846</td>
			<td></td>
		</tr>
		<tr>
			<td>CD8&#160;</td>
			<td>Biolegend</td>
			<td>100703</td>
			<td></td>
		</tr>
		<tr>
			<td>CD90.2</td>
			<td>Biolegend</td>
			<td>105314</td>
			<td></td>
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			<td>Collagenase D</td>
			<td>Roche</td>
			<td>11088866001</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAse I</td>
			<td>Invitrogen</td>
			<td>18068015</td>
			<td>Specific activity: &#8805;10 000 units/mg&#160;&#160;&#160;</td>
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		<tr>
			<td>Facs ARIA II sorter</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FACS Melody cell sorter</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fc-Block</td>
			<td>Biolegend</td>
			<td>101320</td>
			<td></td>
		</tr>
		<tr>
			<td>Fc&#949;RI</td>
			<td>eBioscience</td>
			<td>13589885</td>
			<td></td>
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			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>26140079</td>
			<td></td>
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		<tr>
			<td>FlowJo</td>
			<td>FlowJo</td>
			<td></td>
			<td>Flow cytometry analysis data software</td>
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			<td>Gr-1</td>
			<td>Tonbo</td>
			<td>305931</td>
			<td></td>
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			<td>Hanks Balanced Salt Solution (HBSS)</td>
			<td>Homemade</td>
			<td></td>
			<td></td>
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		<tr>
			<td>IL-9</td>
			<td>biolegend</td>
			<td>514103</td>
			<td></td>
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			<td>NK1.1&#160;</td>
			<td>Biolegend</td>
			<td>108704</td>
			<td></td>
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			<td>Nylon mesh</td>
			<td>&#160;&#8206; lba</td>
			<td>B07HYHHX5V</td>
			<td></td>
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			<td>OptiPrep Density Gradient Medium</td>
			<td>Sigma</td>
			<td>D1556</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline&#160;</td>
			<td>Homemade</td>
			<td></td>
			<td></td>
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		<tr>
			<td>RPMI</td>
			<td>Gibco</td>
			<td>11875093</td>
			<td></td>
		</tr>
		<tr>
			<td>Siglec F&#160;</td>
			<td>Biolegend</td>
			<td>155512</td>
			<td></td>
		</tr>
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			<td>Streptavidin</td>
			<td>Biolegend</td>
			<td>405206</td>
			<td></td>
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		<tr>
			<td>TCR-&#946;&#160;</td>
			<td>Biolegend</td>
			<td>109203</td>
			<td></td>
		</tr>
		<tr>
			<td>TCR-&#946; (PE/Cy7)</td>
			<td>Biolegend</td>
			<td>109222</td>
			<td></td>
		</tr>
		<tr>
			<td>TCR-&#947;&#948;&#160;</td>
			<td>Biolegend</td>
			<td>118103</td>
			<td></td>
		</tr>
		<tr>
			<td>Ter119</td>
			<td>Biolegend</td>
			<td>116204</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricine buffer&#160;</td>
			<td>Homemade</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zombie Aqua Fixable Viability Dye</td>
			<td>Biolegend</td>
			<td>423101</td>
			<td></td>
		</tr>
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a strategy for the study of il-9-producing lymphoid cells in the nippostrongylus brasiliensis infection model

IL-9是一种多效性细胞因子，与各种过程相关，包括抗肿瘤免疫，过敏病理诱导和对蠕虫感染的免疫反应，其中它在寄生虫的排出中起重要作用。在 巴西黑圆线虫 感染的小鼠模型中，IL-9主要由肺、小肠和引流淋巴结中发现的CD4 + T淋巴细胞和先天淋巴细胞产生。鉴于IL-9细胞内染色所涉及的技术困难，以及在感染时从小肠分离造血细胞的复杂性，迫切需要一个全面但直接的方案来分析IL-9在该模型中不同淋巴和非淋巴组织中的表达。此处描述的方案概述了CD4 + T细胞和先天淋巴样细胞在整个感染过程中在肺和小肠（ 巴西奈瑟菌靶向的主要器官）以及纵隔和肠系膜淋巴结中产生的IL-9的动力学。此外，它还详细说明了感染所需的幼虫数量，具体取决于感兴趣的细胞类型和器官。该协议旨在通过提供专注于 巴西猪笼草 感染模型中感兴趣的特定细胞、器官和疾病阶段的机会，协助测定的标准化以节省时间和资源。

小鼠皮下注射200 L3期 巴西猪笼草 幼虫，或用PBS进行假对照。调整了该协议中使用的幼虫数量，以便从肺，淋巴组织和小肠中分离出活细胞，这与以前的报告不同，以前的报告使用更高的蠕虫负荷来检测淋巴组织和肺部中的细胞仅4。在感染后第 0、4、7 和 10 天采集肺、纵隔淋巴结、肠系膜淋巴结和小肠，用于淋巴细胞分离和 IL-9 生成人群的特征。在两个或多个独立实验中?...

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A Strategy for the Study of IL-9-Producing Lymphoid Cells in the Nippostrongylus brasiliensis Infection Model

表达IL-9的T和ILC2细胞在 巴西猪笼草 感染期间被诱导，但由于它们的低频率和差异动力学，它们的表征在感染的肠道中在很大程度上被忽视了。该协议描述了从不同靶器官中分离这些细胞，并通过不同感染阶段的流式细胞术 确认 其身份。

巴西黑线虫感染模型中产生IL-9的淋巴细胞研究策略

IL-9 is a pleiotropic cytokine associated with various processes, including antitumor immunity, induction of allergic pathologies, and the immune response ...

a-strategy-for-study-il-9-producing-lymphoid-cells-nippostrongylus

Research

JoVE Journal

Immunology and Infection

A Strategy for the Study of IL-9-Producing Lymphoid Cells in the Nippostrongylus brasiliensis Infection Model

1.2K 次观看.  Universidad Nacional Autónoma de México. 表达IL-9的T和ILC2细胞在 巴西猪笼草 感染期间被诱导，但由于它们的低频率和差异动力学，它们的表征在感染的肠道中在很大程度上被忽视了。该协议描述了从不同靶器官中分离这些细胞，并通过不同感染阶段的流式细胞术 确认 其身份。

视频: A Strategy for the Study of IL-9-Producing Lymphoid Cells in the Nippostrongylus brasiliensis Infection Model

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

在细菌粘附的研究剪应力

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

科研

免疫与感染

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

在细胞命运<em&gt; E.大肠杆菌</em感染后的噬菌体λ

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Cutaneous Leishmaniasis in the Dorsal Skin of Hamsters: a Useful Model for the Screening of Antileishmanial Drugs

Traditionally, hamsters are experimentally inoculated in the snout or the footpad. However in these sites an ulcer not always occurs, measurement of lesion size is a hard procedure and animals show difficulty to eat, breathe and move because of the lesion. In order to optimize the hamster model for cutaneous leishmaniasis, young adult male and female golden hamsters (Mesocricetus auratus) were injected intradermally at the dorsal skin with 1 to 1.5 x l07 promastigotes of Leishmania species and progression of subsequent lesions were evaluated for up to 16 weeks post infection. The golden hamster was selected because it is considered the adequate bio-model to evaluate drugs against Leishmania as they are susceptible to infection by different species. Cutaneous infection of hamsters results in chronic but controlled lesions, and a clinical evolution with signs similar to those observed in humans. Therefore, the establishment of the extent of infection by measuring the size of the lesion according to the area of indurations and ulcers is feasible. This approach has proven its versatility and easy management during inoculation, follow up and characterization of typical lesions (ulcers), application of treatments through different ways and obtaining of clinical samples after different treatments. By using this method the quality of animal life regarding locomotion, search for food and water, play and social activities is also preserved.

Optimization of the experimental hamster model for cutaneous leishmaniasis by intradermal injection of Leishmania promastigotes at the dorsal skin. This approach is useful during inoculation, follow-up, characterization of lesions, application of treatments and obtaining of clinical samples. Locomotion, search for food and water, play and social activities are preserved.

在仓鼠的背部皮肤:皮肤利什曼病Antileishmanial药物筛选的一个有用的模型

Traditionally, hamsters are experimentally inoculated in the snout or the footpad. However in these sites an ulcer not always occurs, measurement of ...

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

移植物抗宿主病的诱导和<em在体内</em&gt; T细胞监测中的应用MHC匹配的小鼠模型

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. However, isolating bacterial RNA from infected cells or tissues is a challenging task, since mammalian RNA mostly dominates the lysates of infected cells. Here we describe an optimized method for RNA isolation of Listeria monocytogenes bacteria growing within bone marrow derived macrophage cells. Upon infection, cells are mildly lysed and rapidly filtered to discard most of the host proteins and RNA, while retaining intact bacteria. Next, bacterial RNA is isolated using hot phenol-SDS extraction followed by DNase treatment. The extracted RNA is suitable for gene transcription analysis by multiple techniques. This method is successfully employed in our studies of Listeria monocytogenes gene regulation during infection of macrophage cells 1-4. The protocol can be easily modified to study other bacterial pathogens and cell types.

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Here we describe a method for bacterial RNA isolation from Listeria monocytogenes bacteria growing inside murine macrophages. This technique can be used with other intracellular pathogens and mammalian host cells.

RNA纯化在细胞内生长<em&gt;李斯特菌</em&gt;巨噬细胞

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. ...

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant viruses may be potentially pathogenic for many species of protists. They belong to the recently described order of Megavirales. The new lineage Faustovirus that has been isolated from sewage samples is distantly related to the mammalian pathogen African swine fever virus. This virus is also specific to its amoebal host, Vermamoeba vermiformis, a protist common in health care water systems. It is crucial to continue isolating new Faustovirus genotypes in order to enlarge its genotype collection and study its pan-genome. We developed new strategies for the isolation of additional strains by improving the use of antibiotic and antifungal combinations in order to avoid bacterial and fungal contaminations of the amoeba co-culture and favoring the virus multiplication. We also implemented a new starvation medium to maintain V. vermiformis in optimal conditions for viruses co-culture. Finally, we used flow cytometry rather than microscopic observation, which is time-consuming, to detect the cytopathogenic effect. We obtained two isolates from sewage samples, proving the efficiency of this method and thus widening the collection of Faustoviruses, to better understand their environment, host specificity and genetic content.

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

We describe here the latest advances in viral isolation for the characterization of new genotypes of Faustovirus, a new asfarvirus-related lineage of giant viruses. This protocol can be applied to the high throughput isolation of viruses, especially giant viruses infecting amoeba.

一种快速战略新Faustoviruses从环境样品使用隔离<em&gt; Vermamoeba vermiformis</em

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant ...

High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In response to these damages, complex molecular machineries rapidly reseal the plasma membrane to restore its barrier function and maintain cell survival. Despite 60 years of research in this field, we still lack a thorough understanding of the cell resealing machinery. With the goal of identifying cellular components that control plasma membrane resealing or drugs that can improve resealing, we have developed a fluorescence-based high-throughput assay that measures the plasma membrane resealing efficiency in mammalian cells cultured in microplates. As a model system for plasma membrane damage, cells are exposed to the bacterial pore-forming toxin listeriolysin O (LLO), which forms large 30-50 nm diameter proteinaceous pores in cholesterol-containing membranes. The use of a temperature-controlled multi-mode microplate reader allows for rapid and sensitive spectrofluorometric measurements in combination with brightfield and fluorescence microscopy imaging of living cells. Kinetic analysis of the fluorescence intensity emitted by a membrane impermeant nucleic acid-binding fluorochrome reflects the extent of membrane wounding and resealing at the cell population level, allowing for the calculation of the cell resealing efficiency. Fluorescence microscopy imaging allows for the enumeration of cells, which constitutively express a fluorescent chimera of the nuclear protein histone 2B, in each well of the microplate to account for potential variations in their number and allows for eventual identification of distinct cell populations. This high-throughput assay is a powerful tool expected to expand our understanding of membrane repair mechanisms via screening for host genes or exogenously added compounds that control plasma membrane resealing.

Here we describe a high-throughput fluorescence-based assay that measures the plasma membrane resealing efficiency through fluorometric and imaging analyses in living cells. This assay can be used for screening drugs or target genes that regulate plasma membrane resealing in mammalian cells.

哺乳动物细胞血浆膜检测效率的高通量测量

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In ...

The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis

Candida species are common fungal commensals of humans colonizing the skin, mucosal surfaces, and gastrointestinal tract. Under certain conditions, Candida can overgrow their natural niches resulting in debilitating mucosal infections as well as life-threatening systemic infections, which are a major focus of investigation due to their associated high mortality rates. Animal models of disseminated infection exist for studying disease progression and dissecting the characteristics of Candida pathogenicity. Of these, the Galleria mellonella waxworm infection model provides a cost-effective experimental tool for high-throughput investigations of systemic virulence. Many other bacterial and eukaryotic infectious agents have been effectively studied in G. mellonella to understand pathogenicity, making it a widely accepted model system. Yet, variation in the method used to infect G. mellonella can alter phenotypic outcomes and complicate interpretation of the results. Here, we outline the benefits and drawbacks of the waxworm model to study systemic Candida pathogenesis and detail an approach to improve reproducibility. Our results highlight the range of mortality kinetics in G. mellonella and describe the variables which can modulate these kinetics. Ultimately, this method stands as an ethical, rapid, and cost-effective approach to study virulence in a model of disseminated candidiasis.

The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis

Galleria mellonella serves as an invertebrate model for disseminated candidiasis. Here, we detail the infection protocol and provide supporting data for the model&#39;s effectiveness.

弥散性念珠菌感染模型

Candida species are common fungal commensals of humans colonizing the skin, mucosal surfaces, and gastrointestinal tract. Under certain conditions, ...

Use of the Invertebrate Galleria mellonella as an Infection Model to Study the Mycobacterium tuberculosis Complex

Tuberculosis is the leading global cause of infectious disease mortality and roughly a quarter of the world&#8217;s population is believed to be infected with Mycobacterium tuberculosis. Despite decades of research, many of the mechanisms behind the success of M. tuberculosis as a pathogenic organism remain to be investigated, and the development of safer, more effective antimycobacterial drugs are urgently needed to tackle the rise and spread of drug resistant tuberculosis. However, the progression of tuberculosis research is bottlenecked by traditional mammalian infection models that are expensive, time consuming, and ethically challenging. Previously we established the larvae of the insect Galleria mellonella (greater wax moth) as a novel, reproducible, low cost, high-throughput and ethically acceptable infection model for members of the M. tuberculosis complex. Here we describe the maintenance, preparation, and infection of G. mellonella with bioluminescent Mycobacterium bovis BCG lux. Using this infection model, mycobacterial dose dependent virulence can be observed, and a rapid readout of in vivo mycobacterial burden using bioluminescence measurements is easily achievable and reproducible. Although limitations exist, such as the lack of a fully annotated genome for transcriptomic analysis, ontological analysis against genetically similar insects can be carried out. As a low cost, rapid, and ethically acceptable model for tuberculosis, G. mellonella can be used as a pre-screen to determine drug efficacy and toxicity, and to determine comparative mycobacterial virulence prior to the use of conventional mammalian models. The use of the G. mellonella-mycobacteria model will lead to a reduction in the substantial number of animals currently used in tuberculosis research.

Use of the Invertebrate Galleria mellonella as an Infection Model to Study the Mycobacterium tuberculosis Complex

Galleria mellonella was recently established as a reproducible, cheap, and ethically acceptable infection model for the Mycobacterium tuberculosis complex. Here we describe and demonstrate the steps taken to establish successful infection of G. mellonella with bioluminescent Mycobacterium bovis BCG lux.

利用无脊椎杆菌作为感染模型研究结核分枝杆菌复合物

Tuberculosis is the leading global cause of infectious disease mortality and roughly a quarter of the world&#8217;s population is believed to be infected ...

Lentiviral CRISPR/Cas9-Mediated Genome Editing for the Study of Hematopoietic Cells in Disease Models

Manipulating genes in hematopoietic stem cells using conventional transgenesis approaches can be time-consuming, expensive, and challenging. Benefiting from advances in genome editing technology and lentivirus-mediated transgene delivery systems, an efficient and economical method is described here that establishes mice in which genes are manipulated specifically in hematopoietic stem cells. Lentiviruses are used to transduce Cas9-expressing lineage-negative bone marrow cells with a guide RNA (gRNA) targeting specific genes and a red fluorescence reporter gene (RFP), then these cells are transplanted into lethally-irradiated C57BL/6 mice. Mice transplanted with lentivirus expressing non-targeting gRNA are used as controls. Engraftment of transduced hematopoietic stem cells are evaluated by flow cytometric analysis of RFP-positive leukocytes of peripheral blood. Using this method, ~90% transduction of myeloid cells and ~70% of lymphoid cells at 4 weeks after transplantation can be achieved. Genomic DNA is isolated from RFP-positive blood cells, and portions of the targeted site DNA are amplified by PCR to validate the genome editing. This protocol provides a high-throughput evaluation of hematopoiesis-regulatory genes and can be extended to a variety of mouse disease models with hematopoietic cell involvement.

Described are protocols for the highly efficient genome editing of murine hematopoietic stem and progenitor cells (HSPC) by the CRISPR/Cas9 system to rapidly develop mouse model systems with hematopoietic system-specific gene modifications.

Lentiviral CRISPR/Cas9介导基因组编辑，用于疾病模型中造血细胞的研究

Manipulating genes in hematopoietic stem cells using conventional transgenesis approaches can be time-consuming, expensive, and challenging. Benefiting ...