这项研究由诺华医学和生物研究基金会 (PYM)、戈特弗里德和朱莉娅 Bangerter Rhyner 基金会 (兆瓦和 PYM) 和大学 (PYM) 的研究池等部分财政支助。额外赠款包括瑞士政府优秀奖学金为外国学者 (对 KAB 和 SM)。我们感谢伊莎贝尔 Fellay 和 Solange Kharoubi 赫斯的技术支持。

根据 Swissethics (swissethics.ch) 的指导原则, 从健康捐赠者的血液中获得人体红细胞。
注意:恶性疟原虫寄生虫培养 (3D7) 和 EV 生产以前是在 Mbagwu ( et al) 中描述的。11由于P. 恶性疟原虫是人类病原体, 请查阅当地的规章以进行处理。文化应该保持不育的整个时间。
1. 电动汽车的荧光标记
注: 以下程序利用标签技术稳定地将绿色荧光染料 (PKH67) 与大脂肪族尾部结合在 EV 膜的脂质区内。反应是在200µL 最终染色量, 其中包含20µM 最终浓度的 PKH67。在室温下执行所有步骤 (20-25 °c)
<ol><li>在离心管中, 将100µg 的电动车放在1毫升的 PBS 中。</li>
	<li>将电动汽车在离心管中以最大速度 (2万 x g) 离心, 以7分钟为颗粒。 注意: 由于 hemozoin 存在于电动车中, 因此应观察到一种红色深褐色颗粒。如果上清是红色的, 那么电动车就有裂解。</li>
	<li>离心后, 小心地吸上清液, 以避免去除任何电动车。 注: 在稀释剂 C 中 resuspending 电动车之前, 尽量减少剩余的缓冲量, 以提高染色的重现性和效率。</li>
	<li>并用重悬100µL 稀释剂 C (2X ev 悬浮) 中的 EV 颗粒, 并轻轻吸管确保完全分散。</li>
	<li>准备一个新的离心管, 并添加4µL 的 PKH67 乙醇染料溶液的100µL 稀释剂 c. 漩涡井混合。在染色前立即准备此2x 染料溶液 (40 µM)。 注意: 为了避免异质染色, 请勿将 PKH67 乙醇染料溶液直接添加到 EV 颗粒上。</li>
	<li>快速, 结合100µL 2X EV 悬浮 (步骤 1.4) 与100µL 2X 染料溶液 (步骤 1.5)。然后, 通过吹打的10倍来混合样品。</li>
	<li>孵化的 EV/染料悬浮从步骤1.6 为5分钟保护, 从光和混合在5分钟孵化期间涡流3-4 倍。 注: 孵化的 EV/染料悬浮较长时间提供没有好处, 因为染色是如此之快。</li>
	<li>通过添加等量 (200 µL) 的血清和孵育1分钟来阻止染色反应, 从而使过量染料具有约束力。 注: 在停止染色前, 用 2万 x g 离心法从电动车上消耗20分钟的血清。</li>
	<li>用 pbs 清洗3次, 在 2万 x g 处旋转5分钟并用重悬电动车在µL 的100个, 达到最终浓度1µg/µL。</li>
</ol>2. 用共焦显微镜可视化电动汽车的吸收
注意: 下面的协议描述了在玻璃盖玻片上生长的内皮细胞对 EV 内化的跟踪。内皮细胞是半永生化的人骨髓内皮细胞, 如12所述。
<ol><li>在无菌环境中工作, 在室温下 (RT), 涂盖玻片0.01% 多聚 l-赖氨酸, 10 分钟以上。</li>
	<li>吸入聚 l-赖氨酸溶液, 使盖玻片完全干燥。</li>
	<li>使用台盼蓝排除和 hemocytometer 进行可行的内皮细胞计数。</li>
	<li>将 1 x 105内皮细胞移植到1毫升完整的内皮细胞生长培养基中, 其中含有补充剂与聚 l-赖氨酸涂层盖玻片, 并让细胞生长成单层。 注意: 培养基中含有生长因子, 允许细胞形成单层, 形成紧密的连接。</li>
	<li>一旦细胞形成单层, 仔细吸入培养基, 并添加1毫升的新鲜培养基来洗涤细胞。再重复一次洗涤。添加50µg PKH67-labeled 电动车和孵育4小时。 注意: 为了找到最佳的摄取时间点, 可以执行一个时间过程。</li>
	<li>4小时后, 吸入培养基, 用 PBS 冲洗细胞3次以去除多余和未绑定的电动车。将3% 多聚甲醛溶液的细胞固定在 PBS 中15分钟。 注: 甲醇可在固定过程中扰乱肌动蛋白;因此, 最好避免含有护或其他溶剂型护的甲醇。建议使用无甲醇甲醛作为固定剂。</li>
	<li>用 PBS 冲洗细胞3次。仔细添加250µL 0.1% 海卫 X-100 在 PBS permeabilize 细胞。在 RT 孵育5分钟。</li>
	<li>用 PBS 洗3次。在 RT 中添加400个阻塞缓冲区 (PBS 中的 5% BSA) µL, 以阻止非特定绑定。</li>
	<li>用 PBS 洗一次细胞。在200µL PBS/1%bsa 每套µL 中稀释5的罗丹明溶液, 以制备染色溶液。吸管200µL 的染色溶液在盖玻片和孵化20分钟的 RT。</li>
	<li>用 PBS 洗3次。Counterstain DNA 为三十年代与赫斯特33342在最后集中2µg 或 mL 在200µL PBS。</li>
	<li>通过添加400µL 的 PBS 来洗涤2x 的盖玻片。小心地抓住盖玻片与镊子, 并倒置它在一滴 antifade 安装介质上的玻璃幻灯片。用纸巾轻轻地取出多余的介质, 并用指甲油封住两侧。</li>
	<li>将受保护的幻灯片存储在4摄氏度的光线下。</li>
	<li>用共焦显微镜在激光激发405、488和 543 nm 和63X、1.3 NA 油目标上可视化细胞, 其荧光量为 450-470 nm (蓝色)、505-530 nm (绿色) 和 620 nm。 注意: 典型的 F 肌动蛋白染色结果显示在图 1中。</li>
</ol>3. 内皮细胞通透性
注: 通过测量内皮细胞单层中罗丹明 B 异硫氰酸酯-葡聚糖 (平均兆瓦 7万) 的转移, 评估内皮细胞通透性。葡聚糖为研究血管通透性提供了极好的工具。
<ol><li>使用0.4% 台盼蓝排斥和 hemocytometer 计数可行的内皮细胞。</li>
	<li>将内皮细胞密度为 0.6 x 105细胞以汇合24井组织培养插入物 (0.4 µm 孔径), 不受干扰地留在至少5天内形成分化的单分子膜。每隔一天刷新一次介质。</li>
	<li>一旦细胞达到单层, 载入电动车 (50 µg 在1毫升) 在上部室。将罗丹明-葡聚糖添加到顶部井中, 最终浓度为20毫克/毫升。孵化细胞在37°c 与 5% CO2。</li>
	<li>通过测量40µL 中整除数的荧光辐射, 监测荧光葡聚糖在井底的转移。设置多标签微板块读取器544毫微米励磁和 590 nm 发射测量。 注: 通过与库存溶液建立的校准曲线, 可以量化扩散葡聚糖的数量。此外, 通过在时间课程中去除外腔介质的小样本 (图 2), 可以进行动力学实验。即使没有任何治疗, 葡聚糖也会通过细胞单层扩散。</li>
</ol>4. 确定嘌呤霉素灵敏度
注意: 在传感细胞系与慢病毒北疆之前, 确定某一特定细胞系的药物的杀死曲线是很重要的。为了确定杀死所有细胞所需的药物浓度的最小量, 用所选药物的增量剂量进行剂量反应实验。由于每个哺乳动物细胞系都有不同的灵敏度, 在试验之前, 应根据以下详细情况确定抗生素的最佳浓度。
<ol><li>使用 hemocytometer 计数单元格。并用重悬内皮细胞浓度为 1 x10 5 细胞/毫升, 在完全内皮细胞生长培养基中含有补充剂组合物。</li>
	<li>板 1 x 104细胞成96井板在最后容量100µL 内皮细胞生长培养基。</li>
	<li>添加100µL 含有抗生素的培养基, 以获得 0.1-10 µg/毫升的浓度 (请参见图 3)。</li>
	<li>使用20X 目标, 每2天在光显微镜下检查细胞的生存能力。培养细胞10-14 天, 并根据细胞生长, 每2-3 天更换含有嘌呤霉素的培养基。</li>
	<li>在标准培养条件下, 将10µL/井 MTS 试剂添加到每个井中, 混合, 并孵育4小时37摄氏度。</li>
	<li>用 490 nm (图 3) 在一个振动筛上简单地摇动盘子, 用平板阅读器测量处理过的和未经治疗的细胞的吸光度。</li>
</ol>5. 慢病毒载体载体对内皮细胞的转导
<ol><li>板 1 x 105内皮细胞在1毫升完全培养基前的前一天转导。当细胞达到50-80% 融合时进行转导。</li>
	<li>在冰上解冻慢病毒北疆, 使整除数的病毒储存避免冻融循环。三十年代在 200 x g 的材料上旋转, 在管子打开之前, 避免溢出。</li>
	<li>在最终浓度为8µg/毫升的细胞中加入铵溴化物。 注: 铵溴有助于转导效率, 但在某些情况下, 它可能是有毒的细胞。因此, 在转导前执行一条杀死曲线来确定最佳浓度。</li>
	<li>轻轻地将盘子搅拌。增加适当数量的病毒微粒 (在 0.5-2 x 106微粒之间) 在适当的传染多样性 (语言) 和旋转板材轻轻地三十年代混合。</li>
	<li>离心机中的细胞-病毒粒子混合在45分钟的 300 x g, 以提高转导效率。在一夜之间孵化出37摄氏度的细胞病毒颗粒混合物。</li>
	<li>小心去除含有病毒的微粒介质, 并适当丢弃。用新鲜的, 预热的完整培养基替换。</li>
	<li>在第二天使用嘌呤霉素启动选定内容: 移除介质并用新鲜介质替换它, 其中包含嘌呤霉素的正确浓度(图 3)。</li>
	<li>收获细胞后嘌呤霉素选择和分离 rna 使用 rna 隔离套件后, 制造商的指示。</li>
	<li>使用反向转录套件对所选 miRNAs 进行 cDNA 合成 (请参阅材料表)。</li>
	<li>根据引用的协议13设置 qPCR 反应。</li>
</ol>

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作者没有什么可透露的。

一些寄生虫, 包括弓形虫、锥、利什曼原虫和滴虫, 触发受感染的宿主细胞释放电动车。根据病原体, 释放的电动车可以调节宿主免疫应答或调解寄生虫之间的细胞通讯6。然而, 几乎没有证据表明这些小囊泡是如何导致疟疾疾病的。在这里, 我们描述了几种方法来调查在疟原虫感染期间 EVs 的功能。例如, 通过用 PKH67 荧光染料对其进行标记, 可以有效地监测接收单元体内</e...

根据世界卫生组织的报告, 2015年全世界有2亿1200万起新的疟疾病例, 大约42.9万人死亡, 主要是五岁以下的儿童1。导致严重疾病的机制, 通常与血管功能障碍有关, 仍未定义为2。疟原虫-iRBCs 分泌小的双脂膜球体, 称为胞外囊泡 (EVs)。众所周知, 这些电动车与感染过程和宿主对感染的免疫反应有潜在的相关性;然而, 对这些小泡在疟疾感染期间的确切功能知之甚少3。他们有可能扮演两个重要的角色: 一方面, 他们可能通过激活巨噬细胞4,5来促进发病机制;另一方面, 他们可能会调解寄生虫之间的细胞通讯和寄生虫和主机6,7。事实上, 寄生虫可以通过电动车相互转移蛋白质或核酸。例如,锥寄生虫罗得西亚 EVs 可以转移毒力因子血清抗性相关 (), 并可针对其他 T . 寄生虫和宿主红细胞 8.此外,恶性疟原虫-iRBCs 通过在电动车内转移核酸来相互通信。这使得寄生虫能够优化和同步其生长。实际上, EVs 可能是 gametocyte 转换的主要调节器, 因此有助于对传输阶段7的调节。
电动车不仅能调节寄生虫, 而且还能调解寄生宿主之间的相互作用。我们最近发现, 电动车从 iRBCs 包含主机衍生的 microRNAs (miRNAs; 小 RNA 物种的范围内21-25 核苷酸9), 由人类内皮细胞占去。在电动车中的 miRNAs 形成一个稳定的复杂与 Ago2 (RNA 诱导沉默复合体的成员), 一旦交付给接受细胞, 能够明确地沉默基因表达和影响细胞的屏障属性10。为研究电动汽车的功能, 开发了标准协议。在这里, 我们首先描述一个协议, 允许电动车的荧光标记, 以调查他们的接收细胞的吸收。此外, 通过使用共焦显微镜, 可以跟踪 EV 的命运在细胞内。几种荧光染料可用于跟踪电动车。胺反应染料, 5 (和-6)-羧基双乙酸琥珀酸酯 (CFSE) 和 Calcein-在囊泡内成为荧光。我们更喜欢使用两亲的标签, PKH, 因为它给一个更明亮和更均匀的信号。此方法为了解 EVs 和接收单元之间的交互提供了重要信息。在某些情况下, 电动车绑定到细胞的表面, 一些囊泡迅速占去。在吸收后, 电动车将他们的货物运送到细胞中, 并在其中发挥调节作用。
在这里, 我们描述了一个协议, 以测量内皮细胞的屏障功能在体外通过量化的转移荧光葡聚糖通过细胞单层。更灵敏的示踪剂可以使用, 如核素标记。但是, 它们需要特殊的安全预防措施才能使用。还有其他的化验方法来测量体外屏障功能, 如 transendothelial 电阻 (志愿者), 它测量紧密接合的完整性。最后可视化 ZO-1, 一个紧密的连接蛋白, 通过免疫荧光允许评估紧密连接完整性以及10。由于 EVs 是复杂和异构实体, 包含若干具有潜在监管特性的货物, 因此过度表达 tshr 一个特定的 RNA 来研究它对接收单元的影响是有用的。因此, 我们还定义了一个协议, 其目的是生成稳定的单元格线, 表达感兴趣的 miRNA10。

<table>
	<tbody>
		<tr>
			<td>PKH67 Green Fluorescent Cell Linker Mini Kit</td>
			<td>Sigma-Aldrich</td>
			<td>MINI67-1KT</td>
			<td></td>
		</tr>
		<tr>
			<td>Diluent C</td>
			<td>Sigma-Aldrich</td>
			<td>G8278</td>
			<td></td>
		</tr>
		<tr>
			<td>poly-L-lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P8920</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>ThermoFisher - Gibco</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Phalloidin CF594</td>
			<td>Biotium</td>
			<td>#00045</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>ThermoFisher</td>
			<td>H3570</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant</td>
			<td>ThermoFisher</td>
			<td>P36934</td>
			<td></td>
		</tr>
		<tr>
			<td>Rhodamine B isothiocyanate&#8211;Dextran</td>
			<td>ThermoFisher</td>
			<td>R9379-250MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Insert with PET membrane transparent Falcon&#160;for plate 24 wells</td>
			<td>Falcon</td>
			<td>353095</td>
			<td></td>
		</tr>
		<tr>
			<td>Endothelial Cell Growth&#160;Medium MV</td>
			<td>Promocell</td>
			<td>C-22020</td>
			<td></td>
		</tr>
		<tr>
			<td>Puromycin dihydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>P9620-10ML</td>
			<td></td>
		</tr>
		<tr>
			<td>MTS Cell Proliferation Colorimetric Assay Kit</td>
			<td>Biovision</td>
			<td>K300-500</td>
			<td></td>
		</tr>
		<tr>
			<td>hexadimethrine bromide</td>
			<td>Sigma-Aldrich</td>
			<td>107689-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>MISSION&#160;Lenti microRNA, Human hsa-miR-451a</td>
			<td>Sigma-Aldrich</td>
			<td>HLMIR0583</td>
			<td></td>
		</tr>
		<tr>
			<td>MISSION Lenti microRNA, ath-miR416, Negative Control 1 Transduction Particles</td>
			<td>Sigma-Aldrich</td>
			<td>NCLMIR001</td>
			<td></td>
		</tr>
		<tr>
			<td>MISSION&#160;Lenti microRNA, Human</td>
			<td>Sigma-Aldrich</td>
			<td>NCLMIR0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica TCS SP5</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>miRNeasy mini Kit</td>
			<td>Qiagen</td>
			<td>217004</td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan MicroRNA Reverse Transcription Kit 1000 reactions</td>
			<td>ThermoFisher</td>
			<td>4366597</td>
			<td></td>
		</tr>
		<tr>
			<td>hsa-mir-451a&#160;RT/750 PCR rxns</td>
			<td>ThermoFisher</td>
			<td>001141</td>
			<td></td>
		</tr>
		<tr>
			<td>U6 snRNA</td>
			<td>ThermoFisher</td>
			<td>001973</td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan Universal Master Mix II, with UNG</td>
			<td>ThermoFisher</td>
			<td>4440038</td>
			<td></td>
		</tr>
		<tr>
			<td>StepOnePlus Real-Time PCR System</td>
			<td>ThermoFisher</td>
			<td>4376600</td>
			<td></td>
		</tr>
	</tbody>
</table>

evaluation of extracellular vesicle function during malaria infection

疟疾是由疟原虫寄生虫引起的危及生命的疾病, 而P. 恶性疟原虫是非洲大陆上最普遍的, 对全球大多数与疟疾有关的死亡负责。包括寄生虫在组织中的封存、血管功能障碍和炎症反应等几个因素影响了疟疾感染者的病情演变。恶性疟原虫-感染的红细胞 (iRBCs) 释放出小的胞外囊泡 (EVs), 其中含有不同种类的货物分子, 它介导寄生虫与宿主之间的致病机制和细胞间通讯。电动汽车是由它们调节其功能的细胞有效地占用的。在这里, 我们讨论的策略, 以解决电动车在寄生虫宿主互动的作用。首先, 我们描述了一种直接的方法来标记和跟踪的 EV 内化的内皮细胞, 使用绿色细胞链接染料。第二, 我们报告了一个简单的方法来测量渗透率跨内皮细胞单层, 使用荧光标记葡聚糖。最后, 我们展示了如何研究小的非编码 RNA 分子在内皮细胞功能中的作用。

在这里, 我们描述了研究电动汽车与宿主细胞相互作用的协议。荧光标记的 EVs 的摄取是通过共焦显微镜 (图 1) 来监测的。内皮细胞有效地占去了电动车, 但是利用 evs 的潜伏期可以优化以跟踪吸收。为了更好地定位电动汽车内部的细胞, 染色肌动蛋白与罗丹明。接下来, 我们使用一个过滤膜的顶端, 其中一层内皮细胞生长。罗丹明 B 异硫氰酸酯-葡聚糖?...

Watch this Scientific Journal Video about Evaluation of Extracellular Vesicle Function During Malaria Infection at JoVE.com

Evaluation of Extracellular Vesicle Function During Malaria Infection

在这项工作中, 我们描述了研究由恶性疟原虫感染的红细胞释放出的细胞外囊 (EVs) 作用的协议。特别是, 我们关注的是电动车与内皮细胞的相互作用。

疟疾感染中胞外囊泡功能的评价

Malaria is a life-threatening disease caused by Plasmodium parasites, with P. falciparum being the most prevalent on the African continent and responsible ...

evaluation-of-extracellular-vesicle-function-during-malaria-infection

Research

JoVE Journal

Immunology and Infection

7.5K 次观看.  University of Fribourg. 在这项工作中, 我们描述了研究由恶性疟原虫感染的红细胞释放出的细胞外囊 (EVs) 作用的协议。特别是, 我们关注的是电动车与内皮细胞的相互作用。

视频: Evaluation of Extracellular Vesicle Function During Malaria Infection

Neutrophil Extracellular Traps: How to Generate and Visualize Them

Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them intracellularly when their antimicrobial granules fuse with the phagosome. We found that neutrophils have an additional way of killing microorganisms: upon activation, they release granule proteins and chromatin that together form extracellular fibers that bind pathogens. These novel structures, or Neutrophil Extracellular Traps (NETs), degrade virulence factors and kill bacteria1, fungi2 and parasites3. The structural backbone of NETs is DNA, and they are quickly degraded in the presence of DNases. Thus, bacteria expressing DNases are more virulent4. Using correlative microscopy combining TEM, SEM, immunofluorescence and live cell imaging techniques, we could show that upon stimulation, the nuclei of neutrophils lose their shape and the eu- and heterochromatin homogenize. Later, the nuclear envelope and the granule membranes disintegrate allowing the mixing of NET components. Finally, the NETs are released as the cell membrane breaks. This cell death program (NETosis) is distinct from apoptosis and necrosis and depends on the generation of Reactive Oxygen Species by NADPH oxidase5. 
Neutrophil extracellular traps are abundant at sites of acute inflammation. NETs appear to be a form of innate immune response that bind microorganisms, prevent them from spreading, and ensure a high local concentration of antimicrobial agents to degrade virulence factors and kill pathogens thus allowing neutrophils to fulfill their antimicrobial function even beyond their life span. There is increasing evidence, however, that NETs are also involved in diseases that range from auto-immune syndromes to infertility6.
We describe methods to isolate Neutrophil Granulocytes from peripheral human blood7 and stimulate them to form NETs. Also we include protocols to visualize the NETs in light and electron microscopy.

Neutrophil Extracellular Traps (NETs) are an important innate immune mechanism to fight pathogenic bacteria, fungi and parasites. Here we describe methods to isolate neutrophil granulocytes from human blood and to activate them to form NETs. We present preparation techniques to visualize NETs in light and electron microscopy.

嗜中性粒细胞胞外的陷阱：如何生成和可视化

Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them ...

科研

免疫与感染

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

Endemic countries are increasingly adopting molecular tools for efficient typing, identification and surveillance against malaria parasites and vector mosquitoes, as an integral part of their control programs1,2,3,4,5. For sustainable establishment of these accurate approaches in operations research to strengthen malaria control and elimination efforts, simple and affordable methods, with parsimonious reagent and equipment requirements are essential6,7,8. Here we present a simple Chelex-based technique for extracting malaria parasite and vector DNA from field collected mosquito specimens.
We morphologically identified 72 Anopheles gambiae sl. from 156 mosquitoes captured by pyrethrum spray catches in sleeping rooms of households within a 2,000 km2 vicinity of the Malaria Institute at Macha. After dissection to separate the head and thorax from the abdomen for all 72 Anopheles gambiae sl. mosquitoes, the two sections were individually placed in 1.5 ml microcentrifuge tubes and submerged in 20 &mu;l of deionized water. Using a sterile pipette tip, each mosquito section was separately homogenized to a uniform suspension in the deionized water. Of the ensuing homogenate from each mosquito section, 10 &mu;l was retained while the other 10 &mu;l was transferred to a separate autoclaved 1.5 ml tube. The separate aliquots were subjected to DNA extraction by either the simplified Chelex or the standard salting out extraction protocol9,10. The salting out protocol is so-called and widely used because it employs high salt concentrations in lieu of hazardous organic solvents (such as phenol and chloroform) for the protein precipitation step during DNA extraction9.
Extracts were used as templates for PCR amplification using primers targeting arthropod mitochondrial nicotinamide adenine dinucleotide dehydrogenase (NADH) subunit 4 gene (ND4) to check DNA quality11, a PCR for identification of Anopheles gambiae sibling species10 and a nested PCR for typing of Plasmodium falciparum infection12. Comparison using DNA quality (ND4) PCR showed 93% sensitivity and 82% specificity for the Chelex approach relative to the established salting out protocol. Corresponding values of sensitivity and specificity were 100% and 78%, respectively, using sibling species identification PCR and 92% and 80%, respectively for P. falciparum detection PCR. There were no significant differences in proportion of samples giving amplicon signal with the Chelex or the regular salting out protocol across all three PCR applications. The Chelex approach required three simple reagents and 37 min to complete, while the salting out protocol entailed 10 different reagents and 2 hr and 47 min' processing time, including an overnight step. Our results show that the Chelex method is comparable to the existing salting out extraction and can be substituted as a simple and sustainable approach in resource-limited settings where a constant reagent supply chain is often difficult to maintain.

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

A rapid and affordable way to extract quality malaria parasite and vector DNA from mosquito specimens is described. Capitalizing on chelating properties of Chelex resin, the simple method enables genotyping of malaria parasites in mosquito mid-gut and salivary gland phases, as well as molecular identification of the Anopheles sibling species by PCR.

一个简单的的Chelex协议对DNA的提取<em&gt;按蚊</em&gt;属。

Endemic countries are increasingly adopting molecular tools for  efficient typing, identification and surveillance against malaria parasites and  vector ...

2D and 3D Chromosome Painting in Malaria Mosquitoes

Fluorescent in situ hybridization (FISH) of whole arm chromosome probes is a robust technique for mapping genomic regions of interest, detecting chromosomal rearrangements, and studying three-dimensional (3D) organization of chromosomes in the cell nucleus. The advent of laser capture microdissection (LCM) and whole genome amplification (WGA) allows obtaining large quantities of DNA from single cells. The increased sensitivity of WGA kits prompted us to develop chromosome paints and to use them for exploring chromosome organization and evolution in non-model organisms. Here, we present a simple method for isolating and amplifying the euchromatic segments of single polytene chromosome arms from ovarian nurse cells of the African malaria mosquito Anopheles gambiae. This procedure provides an efficient platform for obtaining chromosome paints, while reducing the overall risk of introducing foreign DNA to the sample. The use of WGA allows for several rounds of re-amplification, resulting in high quantities of DNA that can be utilized for multiple experiments, including 2D and 3D FISH. We demonstrated that the developed chromosome paints can be successfully used to establish the correspondence between euchromatic portions of polytene and mitotic chromosome arms in An. gambiae. Overall, the union of LCM and single-chromosome WGA provides an efficient tool for creating significant amounts of target DNA for future cytogenetic and genomic studies.

Chromosome painting is a useful method for studying organization of the cell nucleus and evolution of the karyotype. Here, we demonstrate an approach to isolate and amplify specific regions of interest from single polytene chromosomes that are subsequently used for two- and three-dimensional fluorescent in situ hybridization (FISH).

疟疾蚊子中的二维和三维染色体绘画

Fluorescent in situ hybridization (FISH) of whole arm chromosome probes is a robust technique for mapping genomic regions of interest, detecting ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

高通量实时定量RT-PCR检测方法的确定I型和III型干扰素亚型的表达谱

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

Evaluation of Blood Lactate and Plasma Insulin During High-intensity Exercise by Antecubital Vein Catheterization

The measurement of metabolic and endocrinal markers during physical activity is of relevance to understanding the physiological implications of different exercise modalities. During some exercise modalities (e.g., high-intensity interval exercise), blood metabolites and hormonal levels change in short periods of time. In the present study, we describe a method to catheterize the antecubital vein, which allows the collection of several blood samples during exercise. Insulin and venous lactate concentrations were measured during high-intensity exercise by the application of the described method. The exercise consisted of three 30 s bouts of high-intensity exercise separated by 4 min of recovery. After the last recovery period, a Wingate test was performed. Blood samples from the antecubital vein were obtained before and after each 30 s bout and before and after the Wingate test. As a result, it was possible to evaluate the plasma insulin and venous blood lactate variations during the exercise.

Here we present a protocol to obtain several blood samples from the antecubital vein during high-intensity interval training. This protocol may be useful for the measurement of blood metabolites and endocrinal markers during exercise.

Antecubital 静脉插管高强度运动时血乳酸和血浆胰岛素的评价

The measurement of metabolic and endocrinal markers during physical activity is of relevance to understanding the physiological implications of different ...

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Ca2+ is a ubiquitous ion involved in all known cellular processes. While global Ca2+ responses may affect cell fate, local variations in free Ca2+ cytosolic concentrations, linked to release from internal stores or an influx through plasma membrane channels, regulate cortical cell processes. Pathogens that adhere to or invade host cells trigger a reorganization of the actin cytoskeleton underlying the host plasma membrane, which likely affects both global and local Ca2+ signaling. Because these events may occur at low frequencies in a pseudo-stochastic manner over extended kinetics, the analysis of Ca2+ signals induced by pathogens raises major technical challenges that need to be addressed.
Here, we report protocols for the detection of global and local Ca2+ signals upon a Shigella infection of epithelial cells. In these protocols, artefacts linked to a prolonged exposure and photodamage associated with the excitation of Ca2+ fluorescent probes are troubleshot by stringently controlling the acquisition parameters over defined time periods during a Shigella invasion. Procedures are implemented to rigorously analyze the amplitude and frequency of global cytosolic Ca2+ signals during extended infection kinetics using the chemical probe Fluo-4.

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Here, we present protocols to visualize calcium (Ca2+) responses elicited by HeLa cells infected by Shigella. By optimizing the parameters of bacterial infection and imaging with Ca2+ fluorescent probes, atypical global and local Ca2+ signals induced by bacteria over a large range of infection kinetics are characterized.

成像 Ca2 +在志贺菌感染上皮细胞期间的响应

Ca2+ is a ubiquitous ion involved in all known cellular processes. While global Ca2+ responses may affect cell fate, local variations in free Ca2+ ...

Mouse Body Temperature Measurement Using Infrared Thermometer During Passive Systemic Anaphylaxis and Food Allergy Evaluation

Mouse body temperature measurement is of paramount importance for investigating allergies and anaphylactic symptoms. Rectal probes for temperature readings is common, and they have been proven to be accurate and invaluable in this regard. However, this method of temperature measurement requires the mice to be anesthetized in order to insert the probe without injury to the animal. This limits the ability to observe other phenotypes of the mouse simultaneously. In order to investigate other phenotypes while measuring temperatures, rectal probes are not ideal, and another method is desired. Here, we introduce a noninvasive method of temperature measurement that foregoes the requirement for mouse anesthesia while maintaining equal reliability to rectal probes in measuring body temperature. We use an infrared thermometer that detects body surface temperatures at ranges between 2 and 150 mm. This method of body temperature measurement is successful in reliably replicating temperature change trends during passive system anaphylaxis experiments in mice. We show that body surface temperatures are about 2.0 &#176;C lower than rectal probe measurements, but the degree of temperature drop follows the same trend. Furthermore, we use the same technique to observe mice in a food allergy model to evaluate temperature and activity levels simultaneously.

Here we present a new method to accurately measure body temperature differences in passive systemic anaphylaxis (PSA) and food allergy mouse models using an infrared thermometer. This procedure has been accurately duplicated in previous PSA results.

红外测温仪在被动系统过敏反应和食品过敏评价中的应用

Mouse body temperature measurement is of paramount importance for investigating allergies and anaphylactic symptoms. Rectal probes for temperature ...

Automated Image-Based Quantification of Neutrophil Extracellular Traps Using NETQUANT

Neutrophil extracellular traps (NETs) are web-like antimicrobial structures consisting of DNA and granule derived antimicrobial proteins. Immunofluorescence microscopy and image-based quantification methods remain important tools to quantitate neutrophil extracellular trap formation. However, there are key limitations to the immunofluorescence-based methods that are currently available for quantifying NETs. Manual methods of image-based NET quantification are often subjective, prone to error and tedious for users, especially non-experienced users. Also, presently available software options for quantification are either semi-automatic or require training prior to operation. Here, we demonstrate the implementation of an automated immunofluorescence-based image quantification method to evaluate NET formation called NETQUANT. The software is easy to use and has a user-friendly graphical user interface (GUI). It considers biologically relevant parameters such as an increase in the surface area and DNA:NET marker protein ratio, and nuclear deformation to define NET formation. Furthermore, this tool is built as a freely available app, and allows for single-cell resolution quantification and analysis.

Here, we present a protocol for generating neutrophil extracellular traps (NETs) and operating NETQUANT, a fully automatic software option for quantification of NETs in immunofluorescence images.

使用 NETQUANT 对中性粒细胞外陷阱的自动基于图像的定量

Neutrophil extracellular traps (NETs) are web-like antimicrobial structures consisting of DNA and granule derived antimicrobial proteins. ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a striking increase in life expectancy around the globe. Nonetheless, determining vaccine efficacy remains a challenge. Emerging evidence suggests that the current acellular vaccine (aPV) for Bordetella pertussis (B. pertussis) induces suboptimal immunity. Therefore, a major challenge is designing a next-generation vaccine that induces protective immunity without the adverse side effects of a whole-cell vaccine (wPV). Here we describe a protocol that we used to test the efficacy of a promising, novel adjuvant that skews immune responses to a protective Th1/Th17 phenotype and promotes a better clearance of a B. pertussis challenge from the murine respiratory tract. This article describes the protocol for mouse immunization, bacterial inoculation, tissue harvesting, and analysis of immune responses. Using this method, within our model, we have successfully elucidated crucial mechanisms elicited by a promising, next-generation acellular pertussis vaccine. This method can be applied to any infectious disease model in order to determine vaccine efficacy.

Here we present an elegant protocol for in vivo evaluation of vaccine effectiveness and host immune responses. This protocol can be adapted for vaccine models that study viral, bacterial, or parasitic pathogens.

宿主病原学反应及疫苗效果评价

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...

Purification of High Yield Extracellular Vesicle Preparations Away from Virus

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection setting. The presented method is meant to isolate EVs away from virions (i.e., HIV-1), allowing for a higher efficiency and yield compared to conventional ultracentrifugation methods. Our protocol contains three steps: EV precipitation, density gradient separation, and particle capture. Downstream assays (i.e., Western blot, and PCR) can be run directly following particle capture. This method is advantageous over other isolation methods (i.e., ultracentrifugation) as it allows for the use of minimal starting volumes. Furthermore, it is more user friendly than alternative EV isolation methods requiring multiple ultracentrifugation steps. However, the presented method is limited in its scope of functional EV assays as it is difficult to elute intact EVs from our particles. Furthermore, this method is tailored towards a strictly research-based setting and would not be commercially viable.

This protocol isolates extracellular vesicles (EVs) away from virions with high efficiency and yield by incorporating EV precipitation, density gradient ultracentrifugation, and particle capture to allow for a streamlined workflow and a reduction of starting volume requirements, resulting in reproducible preparations for use in all EV research.

高产细胞外囊泡制剂的纯化远离病毒

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection ...