Name: targeting drugs to larval zebrafish macrophages by injecting drug-loaded liposomes
Uploaded: 2020-02-18T13:00:00
Description: Watch this Scientific Journal Video about Targeting Drugs to Larval Zebrafish Macrophages by Injecting Drug-Loaded Liposomes at JoVE.com

这项工作得到了向C.J.H.（新西兰卫生研究理事会和马斯登基金、新西兰皇家学会）和Z.W.（奥克兰大学学院研究发展基金）提供的赠款的支持。作者感谢阿尔哈德·马哈贡卡尔对斑马鱼设施、生物医学成像研究室、奥克兰大学医学院的辅助成像和格雷厄姆·利施克（Graham Lieschke）赠送Tg（mpeg1：EGFP）记者专线的专家管理。

1. 制备药物装的玛丽娜蓝标记脂体
注：携带蓝色荧光染料、Marina Blue 和药物的脂质体使用薄膜水化方法制备，后插入了 poloxamer 188。除非另有规定，否则所有程序均在室温下进行。控制脂体只携带滨海蓝色和PBS。此处的示例描述了使用线粒体靶向抗氧化药物25加载脂体，该药物在代表性结果中用作概念验证。
<ol><li>为了制备脂质体，溶解16.4毫克（22.2μmol）?...

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在这里，我们提供了一个详细的协议，以制定药物加载的脂体，专门针对幼虫斑马鱼的巨噬细胞。该方法可用于剖析巨噬细胞在某些疾病模型中的作用，确保直接有针对性地向巨噬细胞提供药物。此外，当药物的一般毒性限制其使用时，如浸泡等更常规的路线，可以使用。此处描述的协议提供了用于靶向幼虫斑马鱼中先天免疫细胞的其他纳米颗粒系统的替代方案。这些措施包括将抗疟药物利法平?...

单核噬菌体系统为抵御入侵病原体提供了第一道防线。该系统由单核细胞、单核衍生树突细胞和巨噬细胞组成，它们对外来病原体进行积极的噬菌体，从而限制病原体的传播。除了这些噬菌体和微生物效应器功能外，树突状细胞和巨噬细胞还能够产生细胞因子和抗原，以激活适应性免疫系统1。在这些细胞中，巨噬细胞由于功能异质性不同，并参与多种炎症性疾病而受到特别关注，从自身免疫和传染病到癌症2、3、4、5、6、7。巨噬细胞的可塑性及其在功能上适应组织环境需求的能力，需要采用实验方法直接观察和询问体内的这些细胞。
幼虫斑马鱼是研究体内巨噬细胞功能和可塑性的理想模型生物。幼虫斑马鱼的光学透明度为直接观察巨噬细胞的行为提供了一个窗口，特别是当与巨噬细胞标记转基因报告线结合时。利用幼虫斑马鱼的活成像潜力和实验可采性，对与人类疾病9、10、11、12、13、14、15直接相关的巨噬菌体功能提出了许多重要的见解。其中许多研究还利用斑马鱼药物活性的高保护（一个属性，支撑了它们作为整个动物药物发现平台16，17，18），利用化学干预药理作用操纵巨噬细胞功能。迄今为止，这些药理治疗主要通过浸泡（要求药物是水溶性的）或直接微注射免费药物（图1A）来提供。这些被动分娩策略的局限性包括脱靶效应和一般毒性，可能妨碍评估对巨噬菌体功能的任何影响。此外，在调查药物对巨噬细胞的影响时，不知道药物是作用于巨噬细胞本身或通过更间接的机制。在进行类似的化学干预研究以研究巨噬细胞功能时，我们认识到，开发一种廉价而直接的传递方法，专门针对巨噬细胞的药物，是未满足的。
脂质体是微观的，生物相容性，脂质双层囊泡，可以封装蛋白质，核苷酸和药物货物19。脂质体的单层或多层脂质双层结构形成水性内流明，其中水溶性药物可以结合，而疏水性药物可以集成到脂质膜中。此外，脂质体的物理化学性质，包括大小，电荷和表面修改可以操纵，以定制其靶向到特定的细胞20，21。脂体的这些特点，使他们成为一个有吸引力的工具，提供药物，并提高目前的治疗方案20的精度。由于脂质体是由巨噬细胞自然噬菌体（一种利用其常规使用，专门用于向巨噬细胞进行消融实验的结节功能的一种特征22），它们作为巨噬细胞特异性药物输送的一个有吸引力的选择（图1B）。
该协议描述了药物配方成蓝色荧光脂质体涂覆与亲水性聚合物波罗沙默188，在脂质体表面形成保护层，并已证明，以提高药物保留，并具有优越的生物相容性23。Poloxamer被选择用于脂质体的表面涂层，因为我们先前的研究表明，与聚乙烯乙二醇改性脂质体相比，Poloxamer修饰的脂质体在静脉注射大鼠爪和类似的药代动力学后，在兔子的皮下注射后表现出更好的生物相容性23。还介绍了其向幼虫斑马鱼和活成像进行显微注射的规程，以评估其巨噬细胞靶向能力和细胞内腔内的定位，这些细胞是脂质降解和细胞质药物输送所必需的。作为概念验证，我们以前曾使用这种技术来瞄准两种药物，以巨噬细胞来抑制其激活在幼虫斑马鱼模型急性痛风炎症24。这种药物输送技术扩展了斑马鱼研究人员的化学"工具包"，这些研究人员希望确保其感兴趣的药物的巨噬菌体靶向。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)</td>
			<td>Avanti Polar Lipids, Inc.</td>
			<td>850355P</td>
			<td></td>
		</tr>
		<tr>
			<td>1,2-diseteroyl-sn-glycero-3-phosphocholine (DSPE)</td>
			<td>Avanti Polar Lipids, Inc.</td>
			<td>850367P</td>
			<td></td>
		</tr>
		<tr>
			<td>1.0 &#181;m Whatman Nuclepore Track-Etched polycarbonate membranes</td>
			<td>GE Healthcare Life Sciences</td>
			<td>110610</td>
			<td></td>
		</tr>
		<tr>
			<td>25 mL round-bottom flask</td>
			<td>Sigma-Aldrich</td>
			<td>Z278262</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm culture dish</td>
			<td>Thermo Scientific</td>
			<td>150460</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Sigma-Aldrich</td>
			<td>34998</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent 1260 Infinity Diode Array Detector</td>
			<td>Agilent Technologies</td>
			<td>G4212B</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent 1260 Infinity Quaternary Pump</td>
			<td>Agilent Technologies</td>
			<td>G1311B</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent 1290 Infinity Series Thermostat</td>
			<td>Agilent Technologies</td>
			<td>G1330B</td>
			<td></td>
		</tr>
		<tr>
			<td>Avanti mini-extruder Avanti Polar Lipids Inc.</td>
			<td>Avanti Polar Lipids Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>borosilicate microinjection needles</td>
			<td>Warner Instruments</td>
			<td>203-776-0664</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>C4901-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>cholesterol</td>
			<td>Sigma-Aldrich</td>
			<td>C8667</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont No.5 fine tip forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Microloader pipette tip</td>
			<td>Eppendorf</td>
			<td>5242956003</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf SmartBlock 1.5 mL, thermoblock for 24 reaction vessels</td>
			<td>Eppendorf</td>
			<td>4053-6038</td>
			<td></td>
		</tr>
		<tr>
			<td>eyelash manipulator</td>
			<td>Ted Pella Inc.</td>
			<td>113</td>
			<td></td>
		</tr>
		<tr>
			<td>hemocytometer</td>
			<td>Hawksley</td>
			<td>BS.748</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>BDH Chemicals</td>
			<td>441474J</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC system</td>
			<td>Agilent Technologies</td>
			<td>1260 series HPLC system</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P9541-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>low melting point agarose</td>
			<td>Invitrogen</td>
			<td>16520-100</td>
			<td></td>
		</tr>
		<tr>
			<td>LysoTracker Deep Red</td>
			<td>Invitrogen</td>
			<td>L12492</td>
			<td>1 mM stock solution in DMSO, keep at -20 &#176;C and protect from light.</td>
		</tr>
		<tr>
			<td>LysoTracker Deep Red</td>
			<td>Thermo Scientific</td>
			<td>L12492</td>
			<td></td>
		</tr>
		<tr>
			<td>magnetic stand</td>
			<td>Narishige</td>
			<td>GJ-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Marina Blue 1,2-dihexadecanoyl-sn-glycero-phosphoethanolamine (Marina Blue DHPE)</td>
			<td>Invitrogen</td>
			<td>M12652</td>
			<td>Keep at -20 &#176;C and protect from light.</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma-Aldrich</td>
			<td>34860</td>
			<td></td>
		</tr>
		<tr>
			<td>methyl cellulose</td>
			<td>Sigma-Aldrich</td>
			<td>M0387-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>methylene blue</td>
			<td>Alfa Aesar</td>
			<td>42771</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Sigma-Aldrich</td>
			<td>230391-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>micromanipulator</td>
			<td>Narishige</td>
			<td>M-152</td>
			<td></td>
		</tr>
		<tr>
			<td>mineral oil</td>
			<td>Sigma-Aldrich</td>
			<td>M-3516</td>
			<td></td>
		</tr>
		<tr>
			<td>Mitochondria-targeting antioxidant MitoTEMPO</td>
			<td>Sigma-Aldrich</td>
			<td>SML0737</td>
			<td></td>
		</tr>
		<tr>
			<td>MitoSOX Red Mitochondrial Superoxide Indicator</td>
			<td>Thermo Scientific</td>
			<td>M36008</td>
			<td></td>
		</tr>
		<tr>
			<td>MitoTEMPO</td>
			<td>Sigma-Aldrich</td>
			<td>SML0737</td>
			<td>Keep at -20 &#176;C and protect from light.</td>
		</tr>
		<tr>
			<td>N-Phenylthiourea (PTU)</td>
			<td>Sigma-Aldrich</td>
			<td>P7629-10G</td>
			<td>Take care when handling, toxic.</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>BDH Chemicals</td>
			<td>27810.295</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (pH 7.4)</td>
			<td>Gibco</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish (100 mm x 20 mm)</td>
			<td>Corning Inc.</td>
			<td>430167</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenomenex C18 Gemini-NZ 3 mm 250 mm x 4.6 mm column</td>
			<td>Phenomenex</td>
			<td>00G-4439-E0</td>
			<td></td>
		</tr>
		<tr>
			<td>pHrodo Red Escherichia coli BioParticles Conjugate</td>
			<td>Thermo Scientific</td>
			<td>P35361</td>
			<td></td>
		</tr>
		<tr>
			<td>pHrodo Red Escherichia coli BioParticles Conjugate</td>
			<td>Invitrogen</td>
			<td>P35361</td>
			<td>Keep at -20 &#176;C and protect from light. Make 1 mg/mL stock solution by dissolving 2 mg lyophilized product in 2 mL of PBS supplemented with 20 mM HEPES, pH 7.4.</td>
		</tr>
		<tr>
			<td>plastic transfer pipette</td>
			<td>Medi&#39;Ray</td>
			<td>RL200C</td>
			<td></td>
		</tr>
		<tr>
			<td>poloxamer 188</td>
			<td>BASF Corporation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pressure injector</td>
			<td>Applied Scientific Instruments</td>
			<td>MPPI-2</td>
			<td></td>
		</tr>
		<tr>
			<td>rotary evaporator</td>
			<td>B&#252;chi, Flawil, Switzerland</td>
			<td>B&#252;chi R-215 Rotavapor</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning confocal microscope</td>
			<td>Olympus</td>
			<td>Olympus FV1000 FluoView</td>
			<td></td>
		</tr>
		<tr>
			<td>Sorvall WX+ Ultracentrifuge</td>
			<td>Thermo Scientific</td>
			<td>75000090</td>
			<td></td>
		</tr>
		<tr>
			<td>stereomicroscope</td>
			<td>Leica</td>
			<td>MZ12</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricaine</td>
			<td>Sigma-Aldrich</td>
			<td>A5040-25G</td>
			<td>Make 4 mg/mL stock solution (in deionzed H2O) and keep at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>triton-X100</td>
			<td>Sigma-Aldrich</td>
			<td>X100-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic bath</td>
			<td>Thermo Scientific</td>
			<td>FB-11205</td>
			<td></td>
		</tr>
		<tr>
			<td>Volocity Image Analysis Software</td>
			<td>PerkinElmer</td>
			<td>version 6.3</td>
			<td></td>
		</tr>
		<tr>
			<td>water bath</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zetasizer Nano</td>
			<td>Malvern Instruments Ltd</td>
			<td>Zetasizer Nano ZS ZEN 3600</td>
			<td></td>
		</tr>
	</tbody>
</table>

targeting drugs to larval zebrafish macrophages by injecting drug-loaded liposomes

斑马鱼 （Danio rerio） 幼虫已经发展成为一个流行的模型，研究宿主-病原体相互作用和先天免疫细胞对炎症性疾病的贡献，由于其功能保守的先天免疫系统.它们还广泛用于研究先天免疫细胞如何帮助指导发育过程。通过利用幼虫斑马鱼的光学透明度和遗传可采性，这些研究通常侧重于活成像方法，以功能特征表荧光标记巨噬细胞和中性粒细胞在完整的动物。由于其在疾病发病机制中各异质性和不断扩大的作用，巨噬细胞受到广泛关注。除了基因操作，化学干预现在经常被用来操纵和检查幼虫斑马鱼的巨噬细胞行为。这些药物的输送通常仅限于通过直接浸泡或显微注射被动地瞄准免费药物。这些方法依赖于以下假设：对巨噬细胞行为的任何变化都是药物对巨噬细胞本身的直接影响的结果，而不是对另一种细胞类型的直接影响的下游后果。在这里，我们提出我们的方案，通过显微注射药物加载荧脂体，专门针对幼斑马鱼巨噬细胞的药物。我们揭示，波罗夏默188改性药物加载蓝色荧光脂质体很容易被巨噬细胞，而不是中性粒细胞。我们还提供了证据，证明以这种方式交付的药物可以按照药物作用机制的方式影响巨噬菌体活动。这项技术对于研究人员来说，如果能够确保药物针对巨噬细胞，当药物毒性太大，无法通过浸泡等传统方法输送时，这项技术将有价值的。

本文描述的用于制备荧光脂质体封闭药物的薄膜水化方法是一种简单而经济高效的方法。根据本研究所使用的方案，脂体有望成为单层23、24。表1总结了所生产的脂质体的大小、齐塔潜力、药物载荷和诱捕效率。脂体粒体（药物加载前后）的粒径相似（表1）。与对照脂质体相比，药物载脂质体的表面电荷（Zeta电位）略为中性，...

Watch this Scientific Journal Video about Targeting Drugs to Larval Zebrafish Macrophages by Injecting Drug-Loaded Liposomes at JoVE.com

Targeting Drugs to Larval Zebrafish Macrophages by Injecting Drug-Loaded Liposomes

在这里，我们描述了药物加载脂体及其显微注射到幼虫斑马鱼的合成，目的是将药物输送到巨噬细胞。

通过注射药物加载的脂体将药物定位到拉瓦尔斑马鱼巨噬细胞

Zebrafish (Danio rerio) larvae have developed into a popular model to investigate host-pathogen interactions and the contribution of innate immune cells ...

该协议描述了一种将感兴趣的药物对准幼虫斑马鱼巨噬细胞的技术，以操纵和评估巨噬细胞功能。与传统的药物输送策略相比，该技术具有优势，例如通过确保药物通过脂类体封装专门针对巨噬细胞进行浸入式药物。当应用于具有炎症成分的斑马鱼疾病模型时，这种技术有可能解决巨噬细胞如何促进病理过程。在这里，我们演示了这项技术的效用，将线粒体反应性氧种抑制药物靶向巨噬细胞抑制活化。药物加载的脂质体对幼虫的微注射可能具有挑战性，因为应注意不要造成过度的上皮损伤，从而可能影响免疫学研究。该协议的视觉演示很重要，因为它涉及药理学和整个动物活细胞方法，这些方法可能不会在单个实验室中常规进行。要在25毫升的圆底烧瓶中准备10毫克脂质体，在烧瓶中加入31个海洋蓝纳米摩尔和300纳米摩尔的线粒体反应性氧物种抑制药物。短暂声波，以完全溶解，并在真空压力下每分钟75次旋转的旋转蒸发器上缓慢地去除溶剂。当在烧瓶的玻璃壁内形成干脂薄膜时，在氮气下进一步干燥薄膜45分钟，以方便有机溶剂的完全去除。接下来，用一毫升PBS水合薄膜，用石蜡膜密封烧瓶，然后用手在60摄氏度的水浴中搅拌烧瓶20分钟。经过七个循环的冷冻和解冻后，使用微型挤出机和一微米轨道蚀刻聚碳酸酯膜将脂质体挤出两到六个循环。获得大型单核囊泡后，通过超离心将新鲜的脂质体浓缩成颗粒。在具有两毫升微离温管附件的 ThermoMixer 中，用 1%poloxamer 188 溶液的一毫升孵育颗粒，在 45 摄氏度下孵育 30 分钟，每分钟旋转 750 次。在将马球菌188插入脂质体后，正确的马球菌浓度、孵化时间和温度对于实现所所得脂质体的最佳物理化学特性非常重要。在孵育结束时，在相同的离心机条件下再次对混合物进行超离心机，并去除含有任何未绑定聚合物或药物的上流液。然后将脂体颗粒储存在摄氏四度，防止光线。要确定脂质体的大小和泽塔电位，请用超纯水以 1 比 100 的比例稀释脂质体配方。使用动态光散射分析仪测量 25 摄氏度时三倍的颗粒大小、多散射指数和 Zeta 电位。要计算脂质体诱捕效率和药物负荷，请溶解Triton X-100中的脂质体。将 20 微升溶解脂体样品注入高压液相色谱系统中，并配有四元泵、230 纳米的二极管阵列检测器以及三微米、250 倍-4.6 毫米柱。为准备注射转基因斑马鱼幼虫，使用细尖钳手动对受精后30小时的胚胎进行脱毛，并允许胚胎在28.5摄氏度下发育，直到受精后两天。当胚胎达到适当阶段时，在无菌PBS中以一比一的比例稀释新鲜准备的脂体配方，通过涡旋混合溶液两到三秒钟。将足够的 3%甲基纤维素倒入 35 毫米培养小盘盖的盖子中，形成覆盖整个表面的薄膜。使用塑料转移移液器收集大约 20 到 25 个麻醉幼虫的池。允许胚胎通过重力浓缩在移液器的尖端，并小心地将幼虫转移到35毫米培养盘盖的中间，用最少的溶液。使用微胶囊装载机将幼虫的前部朝向注射板顶部，背部朝向微注射针，用于注射到后脑心室，或将幼虫朝向注射板左侧，其腹体朝向微注射针，以注射到鼻窦内。使用微胶囊装载机用大约五微升的脂体注射混合物对注射针进行背填。将针头安装到连接到磁性支架和压力喷油器的微操纵器中，并在立体显微镜下放置设置。使用清洁、细尖钳切割微注射针的尖端，产生直径约 5 微米的开口。将喷油器设置为大约 50 毫秒的脉冲持续时间和每平方英寸的喷射压力约为 40 磅。将一玻油注入位于血细胞计网格线上的矿物油中，以校准要注入的体积。然后调整脉冲持续时间和/或喷射压力，直到波卢斯的直径覆盖最小栅格线的 2 1/2。小心地将脂体注射混合物的一纳米光注入到每个幼虫的后脑心室。注射后，立即将E3培养基加入注射板，并使用塑料转移移液器轻轻搅拌幼虫的甲基纤维素。要在装有水浸透镜的共体显微镜上成像后脑的后脑表面，首先将 35 毫米培养盘填充到 5 毫米的深度，并安装介质，然后让介质聚合。在足够面积的黄鼠伯中挖掘一条小沟，以容纳实验胚胎的蛋黄囊，并使用填充熔融安装介质的塑料转移移液器收集麻醉幼虫。立即将幼虫放入培养皿中，并使用微胶囊式装载机将蛋黄囊腹侧向下定向到挖掘的孔中。保持胚胎在此位置，直到红糖聚合。用E3介质覆盖胚胎，每毫升三分素补充200微克。要对后脑心室进行实时图像，请选择 20 倍目标，然后将共声显微镜设置为 512 x 512 像素和 2.5 倍变焦。设置显微镜参数后，利用双通道成像从后脑最后脑的后脑表面获得 40 到 3 微米的 Z 堆栈，以成像蓝色荧光脂质体和绿色荧光巨噬细胞。获得所有图像后，将文件加载到适当的 3D 图像分析软件程序中。在"测量"选项卡下，将"查找对象"协议拖动到"在此处拖动任务以进行测量"空间。选择 DAPI 通道以突出显示脂质体，然后拖动检查工具通过 Z 节来检测脂质体大噬细胞。要量化单个细胞中脂质体中的荧光强度，请将"剪辑到感兴趣的区域"协议拖动到"此处拖动任务以进行测量"空间，并使用矩形工具在 X、Y 和 Z 维度中感兴趣的单元格周围绘制一个框。然后从"测量"下拉菜单中选择"使测量项目"，以在库窗口中生成测量文件。在该表中，总结了所生产脂质体的大小、泽塔潜力、药物载荷和诱捕效率。正如所观察到的，脂质体颗粒大小与药物加载和没有药物加载相似，尽管药物加载脂质体的表面电荷比仅使用海洋蓝色的控制脂质体略中性。如所证明，海洋蓝标脂质体微注射到后脑心室，导致居民巨噬细胞迅速吸收，注射后三小时即可通过共声显微镜进行量化。然而，很少观察到嗜中性粒细胞含有细胞内脂质体。在单个脂质体中，脂质体在噬菌体中积累，这是脂质体降解和随后药物含量释放到细胞质所必需的。传递到鼻窦，可以通过循环将脂质体送到造血组织居民巨噬细胞。将含有线粒体活性氧种抑制药物的脂质体微注射到后脑心室，可显著抑制脂质体-后脑-居民巨噬细胞内尿酸晶体驱动的单体尿酸反应性氧物种的产生。通过研究全安装原位杂交和中性粒细胞的时态征，可以进一步验证感兴趣的药物对巨噬细胞活化状态的抑制作用。利用这项技术，我们已经能够将药物靶向巨噬细胞，以确认免疫代谢机制驱动其在急性痛风炎症幼虫斑马鱼模型中的激活。该协议的未来适应可以包括执行脂体的表面修饰，以提高巨噬细胞靶向或促进瞄准其他免疫细胞，如嗜中性粒细胞。

Research

JoVE Journal

Immunology and Infection

Humanized Mouse Model to Study Bacterial Infections Targeting the Microvasculature

人源化的小鼠模型来研究细菌感染瞄准微血管

Neisseria meningitidis causes a severe, frequently fatal sepsis when it enters the human blood stream. Infection leads to extensive damage of the blood ...

科研

免疫与感染

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

使用<em&gt;福氏痢疾杆菌</em&gt;研究自噬，细胞骨架相互作用

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote ...

Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

评估隔离肺泡巨噬细胞的激光共聚焦显微镜抗真菌活性

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic ...

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

人THP-1巨噬细胞通过核转高效转染

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. ...

Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection

造型粘膜念珠菌在斑马鱼幼虫通过鳔注射

Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their ...

Proteomic Profiling of Macrophages by 2D Electrophoresis

巨噬细胞通过二维电泳蛋白组学分析

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The ...

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

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

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 ...

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

胞浆的定量与液泡<em&gt;沙门氏菌</em&gt;在原发性巨噬细胞差透

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like ...

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

通过流式细胞仪大鼠肾脏巨噬细胞的表型特征

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...

Using Zebrafish Models of Human Influenza A Virus Infections to Screen Antiviral Drugs and Characterize Host Immune Cell Responses

使用人类流感病毒感染的斑马鱼模型到屏幕抗病毒药物和表征宿主免疫细胞反应

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare ...