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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S., Licona-Limon, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Th9+cells+and+parasitic+inflammation:+Use+of+Nippostrongylus+and+Schistosoma+models.">Th9 cells and parasitic inflammation: Use of Nippostrongylus and Schistosoma models.</a> Methods in Molecular Biology. 1585, 223-245 (2017).</li><li>Lawrance, C. C., Lucas, E. A., Clarke, S. L., Smith, B. J., Kuvibidila, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effects+of+isoflurane+and+CO2+inhalation+on+plasma+levels+of+inflammatory+markers+associated+with+collagen-induced+arthritis+in+DBA+mice.">Differential effects of isoflurane and CO2 inhalation on plasma levels of inflammatory markers associated with collagen-induced arthritis in DBA mice.</a> International Immunopharmacology. 9 (7-8), 807-809 (2009).</li><li>Boivin, G. P., Bottomley, M. A., Schiml, P. A., Goss, L., Grobe, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiologic,+behavioral,+and+histologic+responses+to+various+euthanasia+methods+in+C57BL/6Ntac+male+mice.">Physiologic, behavioral, and histologic responses to various euthanasia methods in C57BL/6Ntac male mice.</a> Journal of the American Association for Laboratory Animal Science. 56 (1), 69-78 (2017).</li><li>old Spring Harbor Protocols. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hank's+balanced+salt+solution+(HBSS)+without+phenol+red.">Hank's balanced salt solution (HBSS) without phenol red.</a> Cold Spring Harbor Protocols. , (2006).</li><li>Bielecki, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin-resident+innate+lymphoid+cells+converge+on+a+pathogenic+effector+state.">Skin-resident innate lymphoid cells converge on a pathogenic effector state.</a> Nature. 592 (7852), 128-132 (2021).</li><li>Sanjabi, S., Mosaheb, M. M., Flavell, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opposing+effects+of+TGF-beta+and+IL-15+cytokines+control+the+number+of+short-lived+effector+CD8++T+cells.">Opposing effects of TGF-beta and IL-15 cytokines control the number of short-lived effector CD8+ T cells.</a> Immunity. 31 (1), 131-144 (2009).</li><li>Liu, H., Li, M., Wang, Y., Piper, J., Jiang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+single-cell+encapsulation+efficiency+and+reliability+through+neutral+buoyancy+of+suspension.">Improving single-cell encapsulation efficiency and reliability through neutral buoyancy of suspension.</a> Micromachines. 11 (1), 94 (2020).</li><li>Huang, Y., 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=IL-25-responsive,+lineage-negative+KLRG1(hi)+cells+are+multipotential+'inflammatory'+type+2+innate+lymphoid+cells.">IL-25-responsive, lineage-negative KLRG1(hi) cells are multipotential 'inflammatory' type 2 innate lymphoid cells.</a> Nature Immunology. 16 (2), 161-169 (2015).</li><li>Huang, Y., 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=S1P-dependent+interorgan+trafficking+of+group+2+innate+lymphoid+cells+supports+host+defense.">S1P-dependent interorgan trafficking of group 2 innate lymphoid cells supports host defense.</a> Science. 359 (6371), 114-119 (2018).</li><li>Flamar, A. L., 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=Interleukin-33+induces+the+enzyme+Tryptophan+hydroxylase+1+to+promote+inflammatory+group+2+innate+lymphoid+cell-mediated+immunity.">Interleukin-33 induces the enzyme Tryptophan hydroxylase 1 to promote inflammatory group 2 innate lymphoid cell-mediated immunity.</a> Immunity. 52 (4), 606-619 (2020).</li><li>Olgu&#237;n-Mart&#237;nez, E., 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=IL-33+and+the+PKA+pathway+regulate+ILC2+populations+expressing+IL-9+and+ST2.">IL-33 and the PKA pathway regulate ILC2 populations expressing IL-9 and ST2.</a> Frontiers in Immunology. 13, 787713 (2022).</li><li>Olguin-Martinez, E., Ruiz-Medina, B. E., Licona-Limon, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-specific+molecular+markers+and+heterogeneity+in+type+2+innate+lymphoid+cells.">Tissue-specific molecular markers and heterogeneity in type 2 innate lymphoid cells.</a> Frontiers in Immunology. 12, 757967 (2021).</li><li>Noelle, R. J., Nowark, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+sources+and+immune+functions+of+interleukin-9.">Cellular sources and immune functions of interleukin-9.</a> Nature Reviews. Immunology. 10 (10), 683-687 (2010).</li></ol>

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。

<table><tbody><tr><td>ACK buffer</td><td>Homemade</td><td></td><td></td></tr><tr><td>Attune Nxt cytometer</td><td>Thermofisher</td><td></td><td></td></tr><tr><td>B220</td><td>Biolegend</td><td>103204</td><td></td></tr><tr><td>CD11b</td><td>Biolegend</td><td>101204</td><td></td></tr><tr><td>CD11c&amp;nbsp;</td><td>Biolegend</td><td>117304</td><td></td></tr><tr><td>CD19&amp;nbsp;</td><td>Biolegend</td><td>115504</td><td></td></tr><tr><td>CD4</td><td>Biolegend</td><td>100404</td><td></td></tr><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&amp;nbsp;</td><td>Biolegend</td><td>100703</td><td></td></tr><tr><td>CD90.2</td><td>Biolegend</td><td>105314</td><td></td></tr><tr><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: &amp;ge;10 000 units/mg&amp;nbsp;&amp;nbsp;&amp;nbsp;</td></tr><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&amp;epsilon;RI</td><td>eBioscience</td><td>13589885</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Gibco</td><td>26140079</td><td></td></tr><tr><td>FlowJo</td><td>FlowJo</td><td></td><td>Flow cytometry analysis data software</td></tr><tr><td>Gr-1</td><td>Tonbo</td><td>305931</td><td></td></tr><tr><td>Hanks Balanced Salt Solution (HBSS)</td><td>Homemade</td><td></td><td></td></tr><tr><td>IL-9</td><td>biolegend</td><td>514103</td><td></td></tr><tr><td>NK1.1&amp;nbsp;</td><td>Biolegend</td><td>108704</td><td></td></tr><tr><td>Nylon mesh</td><td>&amp;nbsp;&amp;lrm; lba</td><td>B07HYHHX5V</td><td></td></tr><tr><td>OptiPrep Density Gradient Medium</td><td>Sigma</td><td>D1556</td><td></td></tr><tr><td>Phosphate-buffered saline&amp;nbsp;</td><td>Homemade</td><td></td><td></td></tr><tr><td>RPMI</td><td>Gibco</td><td>11875093</td><td></td></tr><tr><td>Siglec F&amp;nbsp;</td><td>Biolegend</td><td>155512</td><td></td></tr><tr><td>Streptavidin</td><td>Biolegend</td><td>405206</td><td></td></tr><tr><td>TCR-&amp;beta;&amp;nbsp;</td><td>Biolegend</td><td>109203</td><td></td></tr><tr><td>TCR-&amp;beta; (PE/Cy7)</td><td>Biolegend</td><td>109222</td><td></td></tr><tr><td>TCR-&amp;gamma;&amp;delta;&amp;nbsp;</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&amp;nbsp;</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></tbody></table>

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?...

Watch this Scientific Journal Video about A Strategy for the Study of IL-9-Producing Lymphoid Cells in the Nippostrongylus brasiliensis Infection Model at JoVE.com

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

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

1.3K 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

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide 1. In chronic infection with Schistosoma, the severe pathology, including liver fibrosis and splenomegaly, is caused by the immune response to the parasite eggs rather than the parasite itself 2. Parasite eggs induce a Th2 response characterized by the production of IL-4, IL-5 and IL-13, the alternative activation of macrophages and the recruitment of eosinophils. Here, we describe injection of Schistosoma mansoni eggs as a model to examine parasite-specific Th2 cytokine responses in the lung and draining lymph nodes, the formation of pulmonary granulomas surrounding the egg, and airway inflammation. 
Following intraperitoneal sensitization and intravenous challenge, S. mansoni eggs are transported to the lung via the pulmonary arteries where they are trapped within the lung parenchyma by granulomas composed of lymphocytes, eosinophils and alternatively activated macrophages 3-6. Associated with granuloma formation, inflammation in the broncho-alveolar spaces, expansion of the draining lymph nodes and CD4 T cell activation can be observed. Here we detail the protocol for isolating Schistosoma mansoni eggs from infected livers (modified from 7), sensitizing and challenging mice, and recovering the organs (broncho-alveolar lavage (BAL), lung and draining lymph nodes) for analysis. We also include representative histologic and immunologic data and suggestions for additional immunologic analysis. 
Overall, this method provides an in vivo model to investigate helminth-induced immunologic responses in the lung, which is broadly applicable to the study of Th2 inflammatory diseases including helminth infection, fibrotic diseases, allergic inflammation and asthma. Advantages of this model for the study of type 2 inflammation in the lung include the reproducibility of a potent Th2 inflammatory response in the lung and draining lymph nodes, the ease of assessment of inflammation by histologic examination of the granulomas surrounding the egg, and the potential for long-term storage of the parasite eggs.

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Schistosoma mansoni eggs are potent stimulators of the T helper type 2 (Th2) immune response, characteristic of parasite infection, asthma and allergic inflammation. This protocol utilizes S. mansoni egg injection to generate a CD4 Th2 cytokine-induced inflammatory response in the lung, characterized by lung granuloma formation around the egg, eosinophilia and macrophage alternative activation.

从使用鸡蛋<em&gt;血吸虫</em&gt;作为<em&gt;在体内</em&gt;蠕虫引起的肺部炎症模型

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide 1. In chronic infection with Schistosoma, the severe ...

科研

免疫与感染

Cultivation of Heligmosomoides Polygyrus: An Immunomodulatory Nematode Parasite and its Secreted Products

Heligmosomoides polygyrus (formerly known as Nematospiroides dubius, and also referred to by some as H. bakeri) is a gastrointestinal helminth that employs multiple immunomodulatory mechanisms to establish chronic infection in mice and closely resembles prevalent human helminth infections. H. polygyrus has been studied extensively in the field of helminth-derived immune regulation and has been found to potently suppress experimental models of allergy and autoimmunity (both with active infection and isolated secreted products). The protocol described in this paper outlines management of the H. polygyrus life cycle for consistent production of L3 larvae, recovery of adult parasites, and collection of their excretory-secretory products (HES).

Cultivation of Heligmosomoides Polygyrus: An Immunomodulatory Nematode Parasite and its Secreted Products

Heligmosomoides polygyrus is a murine nematode with powerful immunomodulatory capabilities that closely resemble those of highly-prevalent human helminth infection. Here we describe a protocol for the long-term maintenance of the H. polygyrus lifecycle.

种植<em&gt; Heligmosomoides Polygyrus:</em&gt;免疫调节线虫寄生虫及其分泌产物

Heligmosomoides polygyrus (formerly known as Nematospiroides dubius, and also referred to by some as H. bakeri) is a gastrointestinal helminth that ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

先天免疫反应中的斑马鱼幼体型号无创成像<em&gt;链球菌iniae</em&gt;感染

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly motile parasite not only reaches the liver to invade hepatocytes and transform into erythrocyte-infective form. It also migrates into the skin and to the proximal lymph node draining the injection site, where it can be recognized and degraded by resident and/or recruited myeloid cells. Intravital imaging reported the early recruitment of brightly fluorescent Lys-GFP positive leukocytes in the skin and the interactions between sporozoites and CD11c+ cells in the draining lymph node. We present here an efficient procedure to recover, identify and enumerate the myeloid cell subsets that are recruited to the mouse skin and draining lymph node following intradermal injection of immunizing doses of sporozoites in a murine model. Phenotypic characterization using multi-parametric flow cytometry provides a reliable assay to assess early dynamic cellular changes during inflammatory response to Plasmodium infection.

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

We describe here a protocol for isolating myeloid cells from mouse skin and draining lymph node following intradermal injection of Plasmodium sporozoites. Flow cytometry of collected cells provides a reliable assay to characterize the skin and draining lymph node inflammatory response to the parasite.

从老鼠皮肤及引流淋巴结髓细胞分离皮下继免疫减毒活<em&gt;疟原虫</em&gt;孢子

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly ...

Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-&#947; Responses

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been highly informative on the role of innate and adaptive immune cells and specific cytokines in host immunity against intracellular pathogens. Production of IFN-&#947; by innate cells during sublethal infection with L. monocytogenes is important for activating macrophages and early control of the pathogen1-3. In addition, IFN-&#947; production by adaptive memory lymphocytes is important for priming the activation of innate cells upon reinfection4. The L. monocytogenes infection model thus serves as a great tool for investigating whether new therapies that are designed to increase IFN-&#947; production have an impact on IFN-&#947; responses in vivo and have productive biological effects such as increasing bacterial clearance or improving mouse survival from infection. Described here is a basic protocol for how to conduct intraperitoneal infections of C57BL/6J mice with the EGD strain of L. monocytogenes and to measure IFN-&#947; production by NK cells, NKT cells, and adaptive lymphocytes by flow cytometry. In addition, procedures are described to: (1) grow and prepare the bacteria for inoculation, (2) measure bacterial load in the spleen and liver, and (3) measure animal survival to endpoints. Representative data are also provided to illustrate how this infection model can be used to test the effect of specific agents on IFN-&#947; responses to L. monocytogenes and survival of mice from this infection.

This protocol describes how to inoculate C57BL/6J mice with the EGD strain of Listeria monocytogenes (L. monocytogenes) and to measure interferon-&#947; (IFN-&#947;) responses by natural killer (NK) cells, natural killer T (NKT) cells, and adaptive T lymphocytes post-infection. This protocol also describes how to conduct survival studies in mice after infection with a modified LD50 dose of the pathogen.

实验性感染<em&gt;李斯特菌</em&gt;作为研究主机干扰素γ响应模型

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been ...

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the pro-inflammatory capabilities of various factors is challenging and requires terminal procedures, such as bronchoalveolar lavage and the removal of lungs for in situ analysis, precluding longitudinal visualization in the same mouse. Here, lung inflammation is induced through the intratracheal instillation of Pseudomonas aeruginosa culture supernatant (SN) in transiently transgenized mice expressing the luciferase reporter gene under the control of a heterologous IL-8 bovine promoter. Luciferase expression in the lung is monitored by in vivo bioluminescent image (BLI) analysis over a 2.5- to 48-h timeframe following the instillation. The procedure can be repeated multiple times within 2 - 3 months, thus permitting the evaluation of the inflammatory response in the same mice without the need to terminate the animals. This approach permits the monitoring of pro- and anti-inflammatory factors acting in the lung in real time and appears suitable for functional and pharmacological studies.

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

The method described here allows for the visualization of IL-8 promoter-dependent inflammation activation in the lungs of mice through non-invasive bioluminescence imaging (BLI). The same animal can be subjected to BLI multiple times for up to two months from the time of delivery of the luciferase reporter construct.

IL-8瞬时转化的小鼠模型<em&gt;体内</em&gt;长期监测炎症反应

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the ...

Humanized NOD/SCID/IL2r&#947;null (hu-NSG) Mouse Model for HIV Replication and Latency Studies

Ethical regulations and technical challenges for research in human pathology, immunology, and therapeutic development have placed small animal models in high demand. With a close genetic and behavioral resemblance to humans, small animals such as the mouse are good candidates for human disease models, through which human-like symptoms and responses can be recapitulated. Further, the mouse genetic background can be altered to accommodate diverse demands. The NOD/SCID/IL2r&#947;null (NSG) mouse is one of the most widely used immunocompromised mouse strains; it allows engraftment with human hematopoietic stem cells and/or human tissues and the subsequent development of a functional human immune system. This is a critical milestone in understanding the prognosis and pathophysiology of human-specific diseases such as HIV/AIDS and aiding the search for a cure. Herein, we report a detailed protocol for generating a humanized NSG mouse model (hu-NSG) by hematopoietic stem cell transplantation into a radiation-conditioned neonatal NSG mouse. The hu-NSG mouse model shows multi-lineage development of transplanted human stem cells and susceptibility to HIV-1 viral infection. It also recapitulates key biological characteristics in response to combinatorial antiretroviral therapy (cART).

This protocol provides a method to establish humanized mice (hu-NSG) via intrahepatic injection of human hematopoietic stem cells into radiation-conditioned neonatal NSG mice. The hu-NSG mouse is susceptible to HIV infection and combinatorial antiretroviral therapy (cART) and serves as a suitable pathophysiological model for HIV replication and latency investigations.

人性化的 nod/scid/il2γ空 (hu-nsg) 小鼠 hiv 复制和延迟研究模型

Ethical regulations and technical challenges for research in human pathology, immunology, and therapeutic development have placed small animal models in ...

Obtainment of Macrophages from Human Monocytes to Assess Leishmania braziliensis Infection Rate and Innate Host Immune Response

Macrophages are multifunctional cells essential to the immune system function, and the primary host cell in Leishmania braziliensis (Lb) infection. These cells are specialized in microorganism recognition and phagocytosis, but also activate other immune cells and present antigens, as well as promote inflammation and tissue repair. Here, we describe a protocol to obtain mononuclear cells from peripheral blood (PBMC) of healthy donors to separate monocytes that then differentiate into macrophages. These cells can then be infected in vitro at different Lb concentrations to evaluate the ability to control infection, as well as evaluate host cell immune response, which can be measured by several methods. PBMCs were first isolated by centrifuging with Ficoll-Hypaque gradient and then plated to allow monocytes to adhere to culture plates; non-adherent cells were removed by washing. Next, adherent cells were cultured with macrophage-colony stimulating factor (M-CSF) for 7 days to induce macrophage differentiation. We suggest plating 2 x 106&#160;cells per well on 24-well plates in order to obtain 2 x 105&#160;macrophages. Fully differentiated macrophages can then be infected with Lb for 4 or 24 hours. This protocol results in a significant percentage of infected cells, which can be assessed by optical or fluorescence microscopy. In addition to infection index, parasite load can be measured by counting the numbers of parasites inside each cell. Further molecular and functional assays can also be performed in culture supernatants or within the macrophages themselves, which allows this protocol to be applied in a variety of contexts and also adapted to other intracellular parasite species.

<meta charset="utf8"></head><body>从人单核细胞中获得巨噬细胞以评估巴西利什曼原虫感染率和先天宿主免疫反应

This protocol describes the process for obtaining human macrophages from monocytes for infection with Leishmania braziliensis. It also allows researchers to evaluate infection rate and parasite viability, ROS production by fluorescence microscopy, and the production of inflammatory mediators in culture supernatants to investigate macrophage response to infection.

从人单核细胞中获得巨噬细胞以评估 巴西利什曼原虫 感染率和先天宿主免疫反应

Macrophages are multifunctional cells essential to the immune system function, and the primary host cell in Leishmania braziliensis (Lb) infection. These ...

Isolation of Group 2 Innate Lymphoid Cells from Mouse Nasal Mucosa to Detect the Expression of CD226

With abundant research on group 2 innate lymphoid cells (ILC2s) published over the years, ILC2s are widely known to be implicated in regulating various pathological processes, including anti-helminth immunity, tissue repair, thermogenesis, and autoimmune diseases such as asthma and allergic rhinitis (AR). ILC2s permanently reside in peripheral tissues such as the skin, gut, lungs, and nasal cavity; however, there is limited information about their exact functions in nasal mucosal immunity. CD226 is an activating costimulatory molecule, mainly expressed on natural killer (NK) cells, T cells, and inflammatory monocytes. However, whether ILC2s express CD226 or play a role in the pathogenesis of ILC2s-related diseases remains unknown. Here, we established a method to isolate and identify ILC2s from the nasal mucosa and detected CD226 expression on ILC2s obtained from healthy and AR mice. Herein, we describe this protocol for the isolation and identification of ILC2s from mouse nasal mucosa, which will help explore the internal pathological mechanism of immunological disorders in nasal mucosal diseases.

Group 2 innate lymphoid cells (ILC2s), implicated in type 2 inflammation, mainly participate in response to helminth infection, allergic diseases, metabolic homeostasis, and tissue repair. In this study, a procedure to isolate ILC2s from murine nasal mucosa and detect the expression of CD226 is demonstrated.

从小鼠鼻粘膜中分离第2组先天淋巴细胞以检测CD226的表达

With abundant research on group 2 innate lymphoid cells (ILC2s) published over the years, ILC2s are widely known to be implicated in regulating various ...

巴西黑线虫感染模型中产生IL-9的淋巴细胞研究策略

摘要

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从使用鸡蛋

种植

先天免疫反应中的斑马鱼幼体型号无创成像

从老鼠皮肤及引流淋巴结髓细胞分离皮下继免疫减毒活

实验性感染

IL-8瞬时转化的小鼠模型

人性化的 nod/scid/il2γ空 (hu-nsg) 小鼠 hiv 复制和延迟研究模型

从人单核细胞中获得巨噬细胞以评估巴西利什曼原虫感染率和先天宿主免疫反应

从人单核细胞中获得巨噬细胞以评估巴西利什曼原虫感染率和先天宿主免疫反应

从小鼠鼻粘膜中分离第2组先天淋巴细胞以检测CD226的表达

巴西黑线虫感染模型中产生IL-9的淋巴细胞研究策略

摘要

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从使用鸡蛋

种植

先天免疫反应中的斑马鱼幼体型号无创成像

从老鼠皮肤及引流淋巴结髓细胞分离皮下继免疫减毒活

实验性感染

IL-8瞬时转化的小鼠模型

人性化的 nod/scid/il2γ空 (hu-nsg) 小鼠 hiv 复制和延迟研究模型

从人单核细胞中获得巨噬细胞以评估 巴西利什曼原虫 感染率和先天宿主免疫反应

从人单核细胞中获得巨噬细胞以评估 巴西利什曼原虫 感染率和先天宿主免疫反应

从小鼠鼻粘膜中分离第2组先天淋巴细胞以检测CD226的表达

从人单核细胞中获得巨噬细胞以评估巴西利什曼原虫感染率和先天宿主免疫反应

从人单核细胞中获得巨噬细胞以评估巴西利什曼原虫感染率和先天宿主免疫反应