作者感谢彭博慈善基金会对约翰霍普金斯疟疾研究所（JHMRI）的财政支持。如果没有JHMRI昆虫和寄生虫学核心设施提供的专业知识，这项工作是不可能的。

下面描述的血液收集已获得约翰霍普金斯大学机构审查委员会的批准。 恶性疟原虫 在生物安全2级（BSL2）设施的无菌条件下在新鲜红细胞中培养，并谨慎处理生物材料。在涉及血液或血液制品的每个步骤之后，每个塑料器皿或玻璃器皿在正确处理之前，都要在罩内用10%的漂白剂冲洗。
1. 试剂和制备
<ol><li>使用寄生虫分离物 恶性疟原虫NF54（ 见材料表），其在连续培养中可产生感染性配子体长达两个月。 注意:并非所有培养适应的寄生虫系都能产生配子体，低通道NF54分离株最一致。</li>
	<li>O+红细胞:通过加入等体积的RPMI 1640培养基稀释全血，并在室温下在旋转转子中以500×g离心5分钟，减加速设置为0。 小心地除去上清液和浅黄色外套（血浆和填充红细胞之间的白色条带，含有大部分白细胞和血小板），并加入等体积的RPMI。重复洗涤步骤两次，最后洗涤后加入一个沉淀体积的RPMI，以获得50%的血细胞比容，以储存在4°C。 注意:配子体在两周内成熟，这使得用新鲜的红细胞开始培养至关重要，通常在一周内绘制的红细胞效果很好。</li>
	<li>O+ 人血清:将至少6个单位的血清混合在一起，以尽量减少来自不同个体的血清正常变化的影响。使用0.2μm过滤瓶对混合的人血清进行灭菌。根据需要以50 mL或更少的份量等分，并储存在-20°C。 注意:红细胞和血清需要来自 恶性疟原虫 培养的相容血型。</li>
	<li>500x次黄嘌呤（100 mL）溶液:将0.5g次黄嘌呤溶解在100 mL 1 M NaOH中。过滤灭菌并制作5-10 mL等分试样进行储存。库存可以在-20°C下储存长达一年，在4°C下储存长达2周。</li>
	<li>碳酸氢钠（可选）:将7.5克碳酸氢钠溶解在100毫升去离子组织培养级水中，并用0.2μm过滤器过滤灭菌。 注意:该协议使用蜡烛罐方法为恶性疟原虫体外培养提供微亲氧条件，并且不需要碳酸氢钠。然而，如果使用疟疾气体混合物（5%O 2，5%CO2和90%N2）培养寄生虫，则重要的是用0.2%碳酸氢钠补充培养基。</li>
	<li>完整培养基:要制备 500 mL 完整培养基，将 1 mL 次黄嘌呤溶液和 50 mL 混合人血清加入 500 mL RPMI 1640 中。如果使用疟疾气体混合物，则加入15 mL 7.5%碳酸氢钠。将整个培养基储存在4°C，如果完整培养基的颜色从橙色变为粉红色，则丢弃。为避免浪费，请在三天内制作足够完整的培养基以供使用。</li>
	<li>除臭培养基（可选）:通过溶解200mg NaHCO3，5mg次黄嘌呤和100μL黄嘌呤酸（从100mM水中储备）到100mL不完全培养基（RPMI 1640与谷氨酰胺和HEPES）来进行除风或乌根酸盐培养基。 注意:驱虫是雌性按蚊中肠中肠内雄性配子形成的过程，在服用感染配子体的血粉几分钟后。疟原虫的雄性配子体在驱虫事件发生后产生8个雄性配子。在体外，当培养温度降低到室温（RT）时，该过程自发发生，并且可以在培养的成熟配子体中观察到。</li>
	<li>N-乙酰氨基葡萄糖（可选）:在水或不完全介质中制备500mMN-乙酰氨基葡萄糖的储备溶液，等分试样并储存在-20°C。</li>
	<li>NaCl溶液:将0.9g，1.6g和12gNaCl溶解在100mL组织培养级去离子水中，制成0.9%，1.6%和12%NaCl溶液。用0.22μm过滤器过滤灭菌并储存在4°C。</li>
</ol>2. 恶性疟原虫无性期培养
<ol><li>要开始寄生虫培养，请从液氮罐中取出 恶性疟原虫 NF54的低通道冷冻小瓶，并在设置为37°C的水浴中快速解冻。</li>
	<li>将内容物（约1毫升）转移到50毫升无菌离心管中，滴加0.2体积的预热12%NaCl，同时轻轻摇动试管以确保均匀混合。在室温下孵育5分钟，间歇性轻轻摇晃。</li>
	<li>滴加9体积的1.6%NaCl，同时继续温和混合。在室温下以 500×g 离心内容物5分钟，小心地丢弃上清液。滴加9体积的0.9%NaCl，同时确保始终如一地混合寄生虫颗粒。在室温下以 500×g 再次离心5分钟，除去上清液并将寄生虫重悬到5mL完整培养基中。</li>
	<li>将内容物转移到6孔组织培养板的一个孔中，并加入100-200μL包装的红细胞。</li>
	<li>将寄生虫培养物在37°C下孵育在蜡烛罐或模块化培养箱中，用5%O 2，5%CO2和90%N2的特殊气体混合物吹扫。</li>
	<li>每天更换培养基，并在常规更换培养基期间吸入培养物后，通过从沉降的红细胞层抽取几微升来进行薄血涂片来监测生长。</li>
	<li>使用无菌玻璃巴斯德移液器抽取，然后点击载玻片的中心，将一滴培养物转移到载玻片上。将另一个载玻片放在液滴的前面，然后将其向后拉以接触红细胞，以一个动作快速将其向前推，以使红细胞单层变薄。</li>
	<li>将载玻片水平放在晾衣架上，让它风干。通过将无水甲醇滴到涂片上来固定血涂片。让固定的涂片完全干燥，然后小心地倒入水中新鲜稀释的10%Geimsa染色剂，直到血涂片完全覆盖。让细胞染色约15分钟。</li>
	<li>通过在干净的自来水中冲洗载玻片，让载玻片在垂直位置干燥，以洗掉多余的污渍。</li>
	<li>通过使用油浸100x物镜在显微镜上观察薄血涂片来确定寄生虫血症。计算总共至少 500 个红细胞中受感染的红细胞数量，以确定寄生虫血症的百分比。</li>
</ol>3. 恶性疟原虫配子细胞培养
注意:配子体培养物需要两周时间才能产生对蚊子具有传染性的成熟配子体。配子体培养的步骤如图 1所示。 恶性疟原虫 分离株在长期体外培养后通常失去产生配子体的能力13。为确保配子体的质量，培养应从低通道饲养层培养物开始，自解冻后不超过2个月。将培养基预热至37°C，并在设置为38°C的玻片加热器上执行所有程序。 确保配子体培养物不会长时间离开培养箱，以尽量减少温度波动。
<ol><li>使用混合无性阶段饲养者培养物接种配子体培养物（第0天），以0.3-1%寄生虫血症和4%血细胞比容进行接种。</li>
	<li>为了建立六孔配子体培养物，在室温下以 500×g 离心5mL饲养层培养物5分钟。弃去上清液并将沉淀重悬于30mL完整培养基中。</li>
	<li>加入1.2mL填充的红细胞，混合并分配5mL到6孔板的每个孔中，并在37°C的蜡烛罐中孵育。 注意:上述建立的配子细胞培养是基于5%寄生虫血症下的混合无性阶段饲养者培养。</li>
	<li>每天更换培养基15-18天，不添加新鲜血液，通过小心吸入约70-80%的培养物上清液以避免去除血细胞。</li>
	<li>向每个孔中加入5 mL新鲜的完整培养基。更换培养基时，使用血清学移液管缓慢地将培养基添加到孔壁上，以避免干扰沉降的红细胞层。 注意:由于在更换培养基期间将留下1-2 mL培养基，因此在配子体培养第1天后，培养物的总体积将在6-7 mL之间。在调整此方案时，建议每隔一天进行一次血涂片检查，以确保寄生虫健康。进行血涂片通常会耗尽培养物中的细胞数量。为避免这种情况，请绘制一个非常小的体积。</li>
	<li>为了量化成熟配子细胞血症，在第15-18天之间进行血涂片，并计算成熟配子体在细胞总数中的数量。 注意:成熟的配子体可以很容易地识别出它们的经典新月形，具有光滑的圆形末端（图5B）。</li>
	<li>计算至少1，000个红细胞，并计算感染成熟配子体的红细胞的百分比。</li>
	<li>为了量化除疹事件，取200μL配子体培养物，并在预热的管中RT下以 500×g 离心5分钟。将沉淀重悬于20μL除臭介质中，并转移到带有盖玻片的载玻片上。</li>
	<li>在室温下孵育15分钟后，使用10倍物镜在相差模式下开始计数除疹中心。对至少四个字段中的外除中心进行计数，以计算外除事件。 注意:Delves等人早些时候已经描述了Flagelation测定的详细方法10。每个场超过20个驱虫事件被认为适合膜进料测定。</li>
	<li>配子体培养物通常具有低水平的残留无性阶段。如果实验需要纯配子体，用50mM N-乙酰氨基葡萄糖处理培养物。</li>
	<li>从每孔10x储备溶液（5 mL）中加入0.5mL N-乙酰氨基葡萄糖，至少3天，同时更换培养基以清除残留的无性阶段。 注意:N-乙酰氨基葡萄糖也可以阻断性生殖子虫的侵袭，治疗应仅在配子体培养的第7天后开始。</li>
</ol>4. 使用标准膜喂养测定法（SMFA）的蚊子感染
注意:体外生长的配子体可以使用玻璃膜喂食器喂给蚊子。采血装置的设置 如图2所示。如上所述，始终将配子体保持在37°C，以避免在蚊子摄入之前激活。预热塑料器皿，试剂和设备与配子体培养物一起使用至37°C。
<ol><li>在膜喂养前，通过去除糖水6.5小时或过夜，使3-7天大的雌性 按蚊 挨饿。</li>
	<li>使用电动或口操作吸气器将它们转移到覆盖有双层细网织物的纸杯中。或者，在冷藏室或冰箱中击倒蚊子，将它们转移到纸杯中。 注意:一品脱大小的杯子可用于喂养多达100只蚊子。</li>
	<li>在室温下以 500×g 离心洗涤的血液5分钟并丢弃上清液。加入等量新鲜解冻的人血清以重建全血。将血液和血清混合物转移到37°C的水浴中30分钟。</li>
	<li>将配子体培养物转移到预热的15mL塑料管中，并在37°C下以 600×g 离心5分钟。 如上所述，小心地吸出培养物上清液并进行薄血涂片，以确定成熟的配子细胞血症。</li>
	<li>为了使最终的血液进料，使用重组的全血将配子体沉淀稀释至所需浓度。将血液喂养保持在37°C，直到蚊子和玻璃喂食器准备就绪。 注意:成熟的配子体浓度在0.02%至0.3%之间，将提供一致的蚊子感染性。然而，建议通过将不同浓度的蚊子喂食到不同的杯子来优化配子细胞血症。</li>
	<li>确保玻璃膜喂食器有两个开口，一个狭窄的顶部开口用于移液受感染的血液，一个底部开口用于膜附着。将石蜡膜切成正方形，拉伸至均匀厚度，然后粘在喂食器的开口处，为血液喂养创造密封的隔间。</li>
	<li>通过连接每侧的管道，将膜给料器连接到循环水浴上，以允许温水通过膜给料器周围的护套。 注:多个玻璃喂料器可以串联连接，以适应多种喂料条件。</li>
	<li>将所有喂料器连接到水浴后，将其打开并检查是否有任何泄漏。</li>
	<li>将喂食器放在蚊帐的中心，放在膜面朝下的蚊杯上，并用夹子或胶带固定喂食器。确保膜喂食器不向任何一侧倾斜，以便血液喂养均匀分布，并使整个喂食器与杯子上的网紧密接触，以允许蚊子从杯子内部进入。</li>
	<li>将约200-1000μL血液移液到膜喂养器中。 注意:血液喂养的体积将根据蚊子的数量和膜喂养器的大小而变化。对于14毫米玻璃喂食器和50只蚊子，使用200μL血液喂养。</li>
	<li>确保正确装载，并将血液喂养覆盖在石蜡膜上。</li>
	<li>让蚊子喂食约30分钟，并进行间歇性监测。 注意:用嘴轻轻吹蚊子以提供CO2， 这将促进改善血液喂养。</li>
	<li>在寒冷的房间里将它们击倒，以清除未喂养的蚊子。明显检查蚊子腹部是否有凸起和发红，作为新鲜血粉的征兆。或者，使用口操作的吸气器选择性地去除未接触的蚊子。</li>
	<li>将未喂养的蚊子浸泡在70%乙醇中后，丢弃它们，并将喂食的蚊子放回蚊子杯中。</li>
	<li>双笼式蚊杯，并将其转移到为感染人类疟疾寄生虫 恶性疟原虫的蚊子指定的高密闭培养箱中。</li>
	<li>将浸泡在10%蔗糖中的化妆棉放在蚊香杯上，为他们提供糖粉。每隔一天更换一次化妆棉，直到蚊子被解剖。</li>
</ol>5. 蚊子中肠道解剖和卵囊负荷定量
注:中肠解剖示意图 如图3所示。
<ol><li>喂血后7-8天，将蚊杯转移到4°C10分钟以击倒蚊子。</li>
	<li>将蚊子转移到冰上的培养皿中，使用细尖镊子将其解剖。</li>
	<li>将蚊子浸泡在70%乙醇中1-2分钟，以安乐死它们。将PBS添加到培养皿中，以便在所有蚊子被安乐死后洗掉乙醇。 注意:在添加PBS之前，请确保所有蚊子都已死亡。</li>
	<li>将载玻片安装在解剖显微镜上，并在中心移液器100μLPBS。小心地使用镊子将一只蚊子转移到PBS中，并将其余的蚊子留在培养皿中冰上。</li>
	<li>使用细尖镊子，从蚊子的后端握住腹部的第三节，用第二镊子保持胸部和腹部之间的连接处。轻轻拉动腹部，直到中肠完全暴露。</li>
	<li>丢弃其余的蚊子组织，并将中肠转移到含有几滴PBS的干净载玻片中。</li>
	<li>当解剖所需数量的蚊子时，使用移液管小心地取出PBS，并用0.2%的亚铬染色中肠2-5分钟。</li>
	<li>去除载玻片上多余的变色和排列中间图，以便在光学显微镜下轻松可视化它们。</li>
	<li>放置一个盖玻片，并在10倍物镜下计数每个中肠上的卵囊。卵囊染成粉红色，呈圆形（图6C）。</li>
</ol>6. 蚊唾液腺解剖和孢子体负荷定量
注:唾液腺解剖示意图如图 4所示。
<ol><li>喂血后14-18天，将蚊杯置于4°C下10分钟。 注意:重要的是要等到蚊子停止移动。</li>
	<li>在等待蚊子停止移动的同时，准备解剖工具。</li>
	<li>将玻璃板放在解剖显微镜载物台上。在 1 mL 注射器和 9 英寸巴斯德移液管和橡胶灯泡上准备 2 套 25 G 针头。将70%EtOH和解剖培养基（HBSS，L-15或PBS）置于6孔板中。</li>
	<li>在冷室中，将麻醉的蚊子转移到含有70%EtOH的6孔板中。用镊子轻轻移动蚊子，以确保所有蚊子都浸泡过，然后使用镊子洗掉70%的EtOH，将蚊子转移到解剖介质中。</li>
	<li>移动到解剖室，将蚊子放在解剖显微镜台上。</li>
	<li>从蚊子身上除去多余的培养基，在解剖过程中根据需要添加新的培养基。避免蚊子变干，但过多的液体会使解剖更加困难。</li>
	<li>使用2个注射器和25 G针头，握住蚊子胸部和头部，轻轻向上拉动头部以将唾液腺从胸部拉出。</li>
	<li>用针头断开唾液腺与头部和胸部的连接。</li>
	<li>将唾液腺暂时置于玻璃板上的单独培养基液滴中。</li>
	<li>解剖15-20个唾液腺后，使用巴斯德移液管将它们收集在低固定管中。 注意:一旦唾液腺进入巴斯德移液器的宽部，它们就会粘在玻璃杯上，很难将它们取出。</li>
	<li>在台式离心机中通过短脉冲旋转来颗粒唾液腺。除去解剖培养基而不破坏沉淀，并加入100μL新的解剖培养基。</li>
	<li>用小匀浆机研磨唾液腺1分钟，得到孢子体。</li>
	<li>将10μL含有孢子体的解剖培养基置于血细胞计数器上。 注意:根据唾液腺的数量，可能需要在计数前用培养基将孢子体溶液稀释至1:10至1:50。</li>
	<li>计算四个象限中两个象限中的孢子体数量，并计算孢子体/蚊子的数量:</li>
</ol><img alt="Equation 1" src="/files/ftp_upload/61426/61426equ01v3.jpg">
<img alt="Equation 2" src="/files/ftp_upload/61426/61426equ02v3.jpg">
<img alt="Equation 3" src="/files/ftp_upload/61426/61426equ03.jpg">

<ol>
	<li>World Health Organization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=World+Malaria+Report.">World Malaria Report.</a> World Health Organization. , (2018).</li><li>Sinden, R. E., Smalley, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gametocytogenesis+of+Plasmodium+falciparum+in+vitro:+The+cell-cycle.">Gametocytogenesis of Plasmodium falciparum in vitro: The cell-cycle.</a> Parasitology. 79 (2), 277-296 (1979).</li><li>Sinden, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sexual+Development+of+Malarial+Parasites.">Sexual Development of Malarial Parasites.</a> Advances in Parasitology. 22, 153-216 (1983).</li><li>Joice, R., 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=Plasmodium+falciparum+transmission+stages+accumulate+in+the+human+bone+marrow.">Plasmodium falciparum transmission stages accumulate in the human bone marrow.</a> Science Translational Medicine. 6 (244), 5 (2014).</li><li>Abdulsalam, A. H., Sabeeh, N., Bain, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immature+Plasmodium+falciparum+gametocytes+in+bone+marrow.">Immature Plasmodium falciparum gametocytes in bone marrow.</a> American Journal of Hematology. 85 (12), 943 (2010).</li><li>Ghosh, A. K., Jacobs-Lorena, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmodium+sporozoite+invasion+of+the+mosquito+salivary+gland.">Plasmodium sporozoite invasion of the mosquito salivary gland.</a> Current Opinion in Microbiology. 12 (4), 394-400 (2009).</li><li>Bennink, S., Kiesow, M. J., Pradel, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+malaria+parasites+in+the+mosquito+midgut.">The development of malaria parasites in the mosquito midgut.</a> Cellular Microbiology. 18 (7), 905-918 (2016).</li><li>Trager, W., Jenson, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cultivation+of+malarial+parasites.">Cultivation of malarial parasites.</a> Nature. 273 (5664), 621-622 (1978).</li><li>Duffy, S., Loganathan, S., Holleran, J. P., Avery, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+production+of+Plasmodium+falciparum+gametocytes+for+malaria+drug+discovery.">Large-scale production of Plasmodium falciparum gametocytes for malaria drug discovery.</a> Nature Protocols. 11 (5), 976-992 (2016).</li><li>Delves, M. J., 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=Routine+in+vitro+culture+of+P.+Falciparum+gametocytes+to+evaluate+novel+transmission-blocking+interventions.">Routine in vitro culture of P. Falciparum gametocytes to evaluate novel transmission-blocking interventions.</a> Nature Protocols. 11 (9), 1668-1680 (2016).</li><li>Habtewold, T., 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=Streamlined+SMFA+and+mosquito+dark-feeding+regime+significantly+improve+malaria+transmission-blocking+assay+robustness+and+sensitivity.">Streamlined SMFA and mosquito dark-feeding regime significantly improve malaria transmission-blocking assay robustness and sensitivity.</a> Malaria Journal. 18 (1), 24 (2019).</li><li>Demanga, C. G., 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=The+development+of+sexual+stage+malaria+gametocytes+in+a+Wave+Bioreactor.">The development of sexual stage malaria gametocytes in a Wave Bioreactor.</a> Parasites and Vectors. 10 (1), 216 (2017).</li><li>Brockelman, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditions+favoring+gametocytogenesis+in+the+continuous+culture+of+Plasmodium+falciparum.">Conditions favoring gametocytogenesis in the continuous culture of Plasmodium falciparum.</a> Journal of Eukaryotic Microbiology. 29, 454-458 (1982).</li><li>Meibalan, E., Marti, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+of+malaria+transmission.">Biology of malaria transmission.</a> Cold Spring Harbor Perspectives in Medicine. 7, (2017).</li><li>Essuman, 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=A+novel+gametocyte+biomarker+for+superior+molecular+detection+of+the+plasmodium+falciparum+infectious+reservoirs.">A novel gametocyte biomarker for superior molecular detection of the plasmodium falciparum infectious reservoirs.</a> Journal of Infectious Diseases. 216 (10), 1264-1272 (2017).</li><li>Sim&#245;es, M. L., Mlambo, G., Tripathi, A., Dong, Y., Dimopoulos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+regulation+of+plasmodium+is+anopheles+species+specific+and+infection+intensity+dependent.">Immune regulation of plasmodium is anopheles species specific and infection intensity dependent.</a> mBio. 8 (5), 01631 (2017).</li><li>Oakley, M. S., 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=Transcriptome+analysis+based+detection+of+Plasmodium+falciparum+development+in+Anopheles+stephensi+mosquitoes.">Transcriptome analysis based detection of Plasmodium falciparum development in Anopheles stephensi mosquitoes.</a> Scientific Reports. 8, 11568 (2018).</li><li>Saraiva, R. G., 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=Chromobacterium+spp.+mediate+their+anti-Plasmodium+activity+through+secretion+of+the+histone+deacetylase+inhibitor+romidepsin.">Chromobacterium spp. mediate their anti-Plasmodium activity through secretion of the histone deacetylase inhibitor romidepsin.</a> Scientific Reports. 8, 6176 (2018).</li><li>Tao, D., 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=Sex-partitioning+of+the+Plasmodium+falciparum+stage+V+gametocyte+proteome+provides+insight+into+falciparum-specific+cell+biology.">Sex-partitioning of the Plasmodium falciparum stage V gametocyte proteome provides insight into falciparum-specific cell biology.</a> Molecular and Cellular Proteomics. 13 (10), 2705-2724 (2014).</li><li>Grabias, B., Zheng, H., Mlambo, G., Tripathi, A. K., Kumar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sensitive+enhanced+chemiluminescent-ELISA+for+the+detection+of+Plasmodium+falciparum+circumsporozoite+antigen+in+midguts+of+Anopheles+stephensi+mosquitoes.">A sensitive enhanced chemiluminescent-ELISA for the detection of Plasmodium falciparum circumsporozoite antigen in midguts of Anopheles stephensi mosquitoes.</a> Journal of Microbiological Methods. 108, 19-24 (2015).</li><li>Ferrer, P., Vega-Rodriguez, J., Tripathi, A. K., Jacobs-Lorena, M., Sullivan, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimalarial+iron+chelator+FBS0701+blocks+transmission+by+Plasmodium+falciparum+gametocyte+activation+inhibition.">Antimalarial iron chelator FBS0701 blocks transmission by Plasmodium falciparum gametocyte activation inhibition.</a> Antimicrobial Agents and Chemotherapy. 59 (3), 1418-1426 (2015).</li><li>Sanders, N. G., Sullivan, D. J., Mlambo, G., Dimopoulos, G., Tripathi, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gametocytocidal+screen+identifies+novel+chemical+classes+with+Plasmodium+falciparum+transmission+blocking+activity.">Gametocytocidal screen identifies novel chemical classes with Plasmodium falciparum transmission blocking activity.</a> PLoS One. 9 (8), 105817 (2014).</li><li>Lindner, S. 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=Transcriptomics+and+proteomics+reveal+two+waves+of+translational+repression+during+the+maturation+of+malaria+parasite+sporozoites.">Transcriptomics and proteomics reveal two waves of translational repression during the maturation of malaria parasite sporozoites.</a> Nature Communications. 10, 4964 (2019).</li><li>McLean, K. J., 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=Generation+of+Transmission-Competent+Human+Malaria+Parasites+with+Chromosomally-Integrated+Fluorescent+Reporters.">Generation of Transmission-Competent Human Malaria Parasites with Chromosomally-Integrated Fluorescent Reporters.</a> Scientific Reports. 9, 13131 (2019).</li><li>Espinosa, D. A., 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=Proteolytic+Cleavage+of+the+Plasmodium+falciparum+Circumsporozoite+Protein+Is+a+Target+of+Protective+Antibodies.">Proteolytic Cleavage of the Plasmodium falciparum Circumsporozoite Protein Is a Target of Protective Antibodies.</a> Journal of Infectious Diseases. 212 (7), 1111-1119 (2015).</li><li>Swearingen, K. 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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+in+vitro+maturation+of+Plasmodium+falciparum+gametocytes.">Complete in vitro maturation of Plasmodium falciparum gametocytes.</a> Nature. 294 (5839), 364-366 (1981).</li><li>Miura, K., 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=An+inter-laboratory+comparison+of+standard+membrane-feeding+assays+for+evaluation+of+malaria+transmission-blocking+vaccines.">An inter-laboratory comparison of standard membrane-feeding assays for evaluation of malaria transmission-blocking vaccines.</a> Malaria Journal. 15, 463 (2016).</li><li>Miura, K., 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=Qualification+of+Standard+Membrane-Feeding+Assay+with+Plasmodium+falciparum+Malaria+and+Potential+Improvements+for+Future+Assays.">Qualification of Standard Membrane-Feeding Assay with Plasmodium falciparum Malaria and Potential Improvements for Future Assays.</a> PLoS One. 8 (3), 57909 (2013).</li></ol>

作者无需透露任何信息。

这里描述的方法已在约翰霍普金斯疟疾研究所成功使用超过10年15、16、17、18、19、20、21、22。使用该协议产生的配子体已被用于高通量配子细胞杀灭测定22，蛋白质组学<sup class="...

疟疾是由 疟原虫 寄生虫引起的，并通过雌性 按 蚊的传染性叮咬传播给脊椎动物宿主。根据2019年世界卫生组织（WHO）的报告，估计有405，000人死亡，而疟疾病例总数为2.28亿例1。大多数与疟疾有关的死亡集中在非洲区域，特别是五岁以下儿童。虽然全球疟疾的总体发病率从2010年开始下降，但近年来下降已趋于平稳，迫切需要采取额外的控制战略来消除这种疾病。
疟疾寄生虫的循环无性血液阶段引起疾病发病机制，其中一小部分分化为雌性和雄性配子体。恶性疟原虫配子体本质上是独一无二的，因为它们需要7-10天才能通过五个形态学上不同的阶段发育。从I期到IV期的未成熟配子体被隔离在骨髓实质中，并且在外周循环中基本上不存在2，3，4，5。感染成熟V期配子体的红细胞在血液中释放并自由循环，被蚊子吸收。一旦进入蚊子中肠，配子体就会被激活，通过温度的变化和暴露于中肠环境，转变为雌性和雄性配子并开始蚊子阶段的发展，其最终达到蚊子唾液腺中孢子体的感染阶段6，7。
由于Trager和Jenson8描述了一种培养恶性疟原虫的标准化方法，对无性血阶段的研究有了很大的进展。然而，由于缺乏可靠的性阶段培养系统，使得研究恶性疟原虫配子体，传播生物学和蚊子阶段变得困难。近年来，已经发表了几种方法，这些方法帮助实验室建立了配子体培养物9，10，11，12。本手稿描述了标准化和可靠的恶性疟原虫配子体培养方案，该协议可以代表疟疾研究界的宝贵资源。这种方法能够稳健地产生成熟和传染性的配子体，再加上标准化的蚊子喂养方案，导致高度可靠的蚊子传染性。建立这些方法是为了维持配子体和蚊子期寄生虫的不间断供应。在这份手稿中，我们描述了一个彻底的配子体培养方案（图1），使用这些膜饲养器制备玻璃膜饲养器和蚊子感染（图2），解剖中肠（图3）和蚊子的唾液腺（图4），以及中肠和唾液腺解剖后蚊子感染的定量。

<table>
	<tbody>
		<tr>
			<td>10% Sugar solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10ml serological pipet</td>
			<td>Falcon</td>
			<td>357551</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml conical tube</td>
			<td>Falcon</td>
			<td>352096</td>
			<td></td>
		</tr>
		<tr>
			<td>1ml serological pipet</td>
			<td>Falcon</td>
			<td>357521</td>
			<td></td>
		</tr>
		<tr>
			<td>25 ml serological pipet</td>
			<td>Falcon</td>
			<td>357535</td>
			<td></td>
		</tr>
		<tr>
			<td>37&#176;C Incubator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml conical tube</td>
			<td>Falcon</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>5ml serological pipet</td>
			<td>Falcon</td>
			<td>357543</td>
			<td></td>
		</tr>
		<tr>
			<td>6 well tissue culture plates</td>
			<td>Falcon</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>70% Ethanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>9&#34; glass pipet</td>
			<td>Fisherbrand</td>
			<td>13-678-6B</td>
			<td></td>
		</tr>
		<tr>
			<td>Anopheles Mosquitoes</td>
			<td>JHMRI, Insectary core</td>
			<td></td>
			<td>We use A. stephensi or A. gambiae (keele)</td>
		</tr>
		<tr>
			<td>cell counter</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Circulating water bath</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>fine tip forceps</td>
			<td>Fisherbrand</td>
			<td>12-000-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Geimsa stain</td>
			<td>Sigma</td>
			<td>GS1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass desiccator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass membrane feeder</td>
			<td>Chemglass Life Sciences</td>
			<td>CG183570</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slides</td>
			<td>Fisherbrand</td>
			<td>12-552-3</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Sigma</td>
			<td>H6648</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Blood O+</td>
			<td>JHU</td>
			<td></td>
			<td>Wash RBCs three times with RPMI and refrigerate at 50% heamatocrit</td>
		</tr>
		<tr>
			<td>Human Serum O+</td>
			<td>Interstate blood bank</td>
			<td></td>
			<td>Pool at-least 6 units of serum from different donors and freeze down aliquots at -20&#176;C.</td>
		</tr>
		<tr>
			<td>Hypoxanthine</td>
			<td>Sigma</td>
			<td>H9337</td>
			<td>Make 500x stock in 1M NaOH</td>
		</tr>
		<tr>
			<td>Mercurochrome</td>
			<td>Sigma</td>
			<td>M7011</td>
			<td>Prepare 1% stock solution in PBS that can be diluted to 0.1% when needed</td>
		</tr>
		<tr>
			<td>Micro Pipette</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus</td>
			<td></td>
			<td>Any microscope with 10x, 40x and 100x objective will work.</td>
		</tr>
		<tr>
			<td>Mosquito cups</td>
			<td>Neptune cups</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>N-acetylglucosamine</td>
			<td>Sigma</td>
			<td>A3286</td>
			<td>Optional and needed only when pure gametocytes are required.</td>
		</tr>
		<tr>
			<td>Netting</td>
			<td></td>
			<td></td>
			<td>Make sure it can contain mosquitoes and allow blood feeding</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Thermo Scientific</td>
			<td>249964</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipetman</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmodium falciparum NF54</td>
			<td>BEI Resources</td>
			<td>MRA-1000</td>
			<td>Freeze down large numbers of early passage culture to make sure you have a constant supply</td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Corning</td>
			<td>CV-041-CV</td>
			<td>Media contains glutamine and HEPES</td>
		</tr>
		<tr>
			<td>Slide warmer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma</td>
			<td>S6297</td>
			<td>Optional for media, add only when using malaria gas mix during culture incubation</td>
		</tr>
		<tr>
			<td>water bath</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Xanthurenic Acid</td>
			<td>Sigma</td>
			<td>D120804</td>
			<td>For flagellation media</td>
		</tr>
	</tbody>
</table>

plasmodium falciparum gametocyte culture and mosquito infection through artificial membrane feeding

疟疾仍然是最重要的公共卫生问题之一，导致严重的发病率和死亡率。疟疾是一种蚊子传播的疾病，通过雌性 按蚊 的传染性叮咬传播。疟疾控制最终将取决于多种方法，其中包括阻断与蚊子之间、通过蚊子传播和传播的方法。为了在实验室中研究疟疾寄生虫的蚊子阶段，我们优化了培养高传染性 恶性疟原 虫配子体的方案，这是从人类宿主传播到蚊子媒介所需的寄生虫阶段。 恶性疟原虫 配子体通过五个形态学上不同的步骤成熟，大约需要1-2周。该协议中描述的配子细胞培养在15天内完成，并且从第15-18天对蚊子具有传染性。这些方案的制定是为了维持感染合格配子体的连续循环，并维持寄生虫蚊子阶段的不间断供应。在这里，我们描述了配子体培养的方法，以及如何使用玻璃膜喂食器用这些寄生虫感染蚊子。

在这里，我们展示了使用使用上述方案生成的 恶性疟原虫 NF54配子体培养物的一系列膜进料的结果（见（图5）。配子体培养在第0天以约0.5%的混合期无性培养开始，到第4天和第5天生长至约15%的寄生虫血症高峰。如图 5A 所示，在这种高度寄生虫血症下，寄生虫受到压力，无性阶段培养崩溃。然而，这种压力同时导致配子细胞生成的诱导。早期配?...

Watch this Scientific Journal Video about Plasmodium falciparum Gametocyte Culture and Mosquito Infection Through Artificial Membrane Feeding at JoVE.com

Plasmodium falciparum Gametocyte Culture and Mosquito Infection Through Artificial Membrane Feeding

对疟疾寄生虫的蚊子阶段进行详细调查对于设计有效的传播阻断策略至关重要。该协议展示了如何有效地培养传染性配子体，然后将这些配子体喂给蚊子以产生 恶性疟原虫的蚊子阶段。

恶性疟原虫 通过人工膜喂养进行配子体培养和蚊子感染

Malaria remains one of the most important public health problems, causing significant morbidity and mortality. Malaria is a mosquito borne disease ...

plasmodium-falciparum-gametocyte-culture-mosquito-infection-through

Research

JoVE Journal

Biology

Plasmodium falciparum Gametocyte Culture and Mosquito Infection Through Artificial Membrane Feeding

7.3K 次观看.  Johns Hopkins University. 对疟疾寄生虫的蚊子阶段进行详细调查对于设计有效的传播阻断策略至关重要。该协议展示了如何有效地培养传染性配子体，然后将这些配子体喂给蚊子以产生 恶性疟原虫的蚊子阶段。

视频: Plasmodium falciparum Gametocyte Culture and Mosquito Infection Through Artificial Membrane Feeding

Harvesting Sperm and Artificial Insemination of Mice

Rodents of the genus Peromyscus (deer mice) are the most prevalent native North American mammals. Peromyscus species are used in a wide range of research including toxicology, epidemiology, ecology, behavioral, and genetic studies. Here they provide a useful model for demonstrations of artificial insemination. 

Methods similar to those displayed here have previously been used in several deer mouse studies, yet no detailed protocol has been published. Here we demonstrate the basic method of artificial insemination. This method entails extracting the testes from the rodent, then isolating the sperm from the epididymis and vas deferens. The mature sperm, now in a milk mixture, are placed in the female&#x2019;s reproductive tract at the time of ovulation. Fertilization is counted as day 0 for timing of embryo development. Embryos can then be retrieved at the desired time-point and manipulated.

Artificial insemination can be used in a variety of rodent species where exact embryo timing is crucial or hard to obtain. This technique is vital for species or strains (including most Peromyscus) which may not mate immediately and/or where mating is hard to assess. In addition, artificial insemination provides exact timing for embryo development either in mapping developmental progress and/or transgenic work. Reduced numbers of animals can be used since fertilization is guaranteed. This method has been vital to furthering the Peromyscus system, and will hopefully benefit others as well.

This protocol demonstrates methods for extracting sperm from the testes of males and then inseminating female mice. This procedure is useful when precise time is needed in developmental studies as well as transgenic work.

收获的小鼠精子和人工授精

Rodents of the genus Peromyscus (deer mice) are the most prevalent native North American mammals.  Peromyscus species are used in a wide range of research ...

科研

生物学

Protocol for Plasmodium falciparum Infections in Mosquitoes and Infection Phenotype Determination

Once a gene is identified as potentially refractory for malaria, it must be evaluated for its role in preventing Plasmodium infections within the mosquito.    This protocol illustrates how the extent of plasmodium infections of mosquitoes can be assayed.  The techniques for preparing the gametocyte culture, membrane feeding mosquitoes human blood, and assaying viral titers in the mosquito midgut are demonstrated.

Once a gene is identified as potentially refractory for malaria, it must be evaluated for its role in preventing Plasmodium infections within the mosquito. This protocol illustrates how the extent of plasmodium infections of mosquitoes can be assayed. The techniques for preparing the gametocyte culture, membrane feeding mosquitoes human blood, and assaying viral titers in the mosquito midgut are demonstrated.

恶性疟原虫在蚊子和病毒感染的表型测定感染的议定书

Once a gene is identified as potentially refractory for malaria, it must be evaluated for its role in preventing Plasmodium infections within the ...

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.

Herein is described the procedure implemented in the Caffrey Membrane Structural and Functional Biology Group to set up manually crystallization trials of membrane proteins in lipidic mesophases.

使用油脂介孔结构的测定结晶膜蛋白

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

使用机器人在油脂中间相高通量膜蛋白的结晶

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at atomic resolution. Presently, there is only one way to capture such information as applied to integral membrane proteins (Figure 1), and the complexes they form, and that method is macromolecular X-ray crystallography (MX). To do MX diffraction quality crystals are needed which, in the case of membrane proteins, do not form readily. A method for crystallizing membrane proteins that involves the use of lipidic mesophases, specifically the cubic and sponge phases1-5, has gained considerable attention of late due to the successes it has had in the G protein-coupled receptor field6-21 (<a href="http://www.mpdb.tcd.ie" target="_blank">www.mpdb.tcd.ie</a>). However, the method, henceforth referred to as the in meso or lipidic cubic phase method, comes with its own technical challenges. These arise, in part, due to the generally viscous and sticky nature of the lipidic mesophase in which the crystals, which are often micro-crystals, grow. Manipulating crystals becomes difficult as a result and particularly so during harvesting22,23. Problems arise too at the step that precedes harvesting which requires that the glass sandwich plates in which the crystals grow (Figure 2)24,25 are opened to expose the mesophase bolus, and the crystals therein, for harvesting, cryo-cooling and eventual X-ray diffraction data collection.
The cubic and sponge mesophase variants (Figure 3) from which crystals must be harvested have profoundly different rheologies4,26. The cubic phase is viscous and sticky akin to a thick toothpaste. By contrast, the sponge phase is more fluid with a distinct tendency to flow. Accordingly, different approaches for opening crystallization wells containing crystals growing in the cubic and the sponge phase are called for as indeed different methods are required for harvesting crystals from the two mesophase types. Protocols for doing just that have been refined and implemented in the Membrane Structural and Functional Biology (MS&amp;FB) Group, and are described in detail in this JoVE article (Figure 4). Examples are given of situations where crystals are successfully harvested and cryo-cooled. We also provide examples of cases where problems arise that lead to the irretrievable loss of crystals and describe how these problems can be avoided. In this article the Viewer is provided with step-by-step instructions for opening glass sandwich crystallization wells, for harvesting and for cryo-cooling crystals of membrane proteins growing in cubic and in sponge phases.

Herein is described procedures implemented in the Caffrey Membrane Structural and Functional Biology Group to harvest and cryo-cool membrane protein crystals grown in lipidic cubic and sponge phases for use in structure determination using macromolecular X-ray crystallography.

在油脂中间相结构的测定生物大分子晶体生长的膜蛋白晶体收获和低温冷

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at ...

Measuring Glutathione-induced Feeding Response in Hydra

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. The movement of prey organisms causes mechanosensory discharge of the stinging cells called nematocysts from hydra, which are inserted into the prey. The feeding response in hydra, which includes curling of the tentacles to bring the prey towards the mouth, opening of the mouth and consequent engulfing of the prey, is triggered by GSH present in the fluid released from the injured prey. To be able to identify the molecular mechanism of the feeding response in hydra which is unknown to date, it is necessary to establish an assay to measure the feeding response. Here, we describe a simple method for the quantitation of the feeding response in which the distance between the apical end of the tentacle and mouth of hydra is measured and the ratio of such distance before and after the addition of GSH is determined. The ratio, called the relative tentacle spread, was found to give a measure of the feeding response. This assay was validated using a starvation model in which starved hydra show an enhanced feeding response in comparison with daily fed hydra.

Here we describe a simple assay for the quantification of the feeding response in hydra induced by the reduced form of glutathione. This assay relies on measuring the distance between the apical end of the tentacle and mouth of hydra.

测量水润谷胱甘肽诱导饲养响应

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. ...

High-fat Feeding Paradigm for Larval Zebrafish: Feeding, Live Imaging, and Quantification of Food Intake

Zebrafish are emerging as a model of dietary lipid processing and metabolic disease. This protocol describes how to feed larval zebrafish a lipid-rich meal, which consists of an emulsion of chicken egg yolk liposomes created by sonicating egg yolk in embryo media. Detailed instructions are provided to screen larvae for egg yolk consumption so that larvae that fail to feed will not confound experimental results. The chicken egg yolk liposomes can be spiked with fluorescent lipid analogs, including fatty acids and cholesterol, enabling both systemic and subcellular visualization of dietary lipid processing. Several methods are described to mount larvae that are conducive to short- and long-term live imaging with both upright and inverted objectives at high and low magnification. Additionally presented is an assay to quantify larval food intake by extracting the lipids of larvae fed fluorescent lipid analogs, spotting the lipids on a thin layer chromatography plate, and quantifying the fluorescence. Finally, critical aspects of the procedures, important controls, options for modifying the protocols to address specific experimental questions, and potential limitations are discussed. These techniques can be applied not only to focused, hypothesis driven inquiries, but also to a variety of screens and live imaging techniques to study dietary lipid metabolism and the control of food intake.

Zebrafish are emerging as a valuable model of dietary lipid processing and metabolic disease. Described are protocols of lipid-rich larval feeds, live imaging of dietary fluorescent lipid analogs, and quantification of food intake. These techniques can be applied to a variety of screening, imaging, and hypothesis driven inquiry techniques.

高脂饮食范式斑马鱼幼体:饲养，实时成像，和食物摄入的量化

Zebrafish are emerging as a model of dietary lipid processing and metabolic disease. This protocol describes how to feed larval zebrafish a lipid-rich ...

Ground State Depletion Super-resolution Imaging in Mammalian Cells

Advances in fluorescent microscopy and cell biology are intimately correlated, with the enhanced ability to visualize cellular events often leading to dramatic leaps in our understanding of how cells function. The development and availability of super-resolution microscopy has considerably extended the limits of optical resolution from ~250-20 nm. Biologists are no longer limited to describing molecular interactions in terms of colocalization within a diffraction limited area, rather it is now possible to visualize the dynamic interactions of individual molecules. Here, we outline a protocol for the visualization and quantification of cellular proteins by ground-state depletion microscopy&#160;for fixed cell imaging. We provide examples from two different membrane proteins, an element of the endoplasmic reticulum translocon, sec61&#946;, and a plasma membrane-localized voltage-gated L-type Ca2+ channel (CaV1.2). Discussed are the specific microscope parameters, fixation methods, photo-switching buffer formulation, and pitfalls and challenges of image processing.

This article outlines a protocol for the detection of one or more plasma and/or intracellular membrane proteins using Ground State Depletion (GSD) super-resolution microscopy in mammalian cells. Here, we discuss the benefits and considerations of using such approaches for the visualization and quantification of cellular proteins.

哺乳动物细胞基态衰竭超分辨成像

Advances in fluorescent microscopy and cell biology are intimately correlated, with the enhanced ability to visualize cellular events often leading to ...

Isolation and Culture of Human Mature Adipocytes Using Membrane Mature Adipocyte Aggregate Cultures (MAAC)

White adipose tissue (WAT) dysregulation plays a central role in development of insulin resistance and type 2 diabetes (T2D). To develop new treatments for T2D, more physiologically relevant in vitro adipocyte models are required. This study describes a new technique to isolate and culture mature human adipocytes. This method is entitled MAAC (membrane mature adipocyte aggregate culture), and compared to other adipocyte in vitro models, MAAC possesses an adipogenic gene signature that is the closest to freshly isolated mature adipocytes. Using MAAC, adipocytes can be cultured from lean and obese patients, different adipose depots, co-cultured with different cell types, and importantly, can be kept in culture for 2 weeks. Functional experiments can also be performed on MAAC including glucose uptake, lipogenesis, and lipolysis. Moreover, MAAC responds robustly to diverse pharmacological agonism and can be used to study adipocyte phenotypic changes, including the transdifferentiation of white adipocytes into brown-like fat cells.

Isolation and Culture of Human Mature Adipocytes Using Membrane Mature Adipocyte Aggregate Cultures MAAC

Membrane mature adipocyte aggregate culture (MAAC) is a new method to culture mature human adipocytes. Here we detail how to isolate adipocytes from human adipose and how to set up MAAC.

利用膜成熟腺细胞聚合培养（MAAC）的人类成熟腺细胞的分离与培养

White adipose tissue (WAT) dysregulation plays a central role in development of insulin resistance and type 2 diabetes (T2D). To develop new treatments ...

Large-Scale Preparation of Synovial Fluid Mesenchymal Stem Cell-Derived Exosomes by 3D Bioreactor Culture

Exosomes secreted by mesenchymal stem cells (MSCs) have been suggested as promising candidates for cartilage injuries and osteoarthritis treatment. Exosomes for clinical application require large-scale production. To this aim, human synovial fluid MSCs (hSF-MSCs) were grown on microcarrier beads, and then cultured in a dynamic three-dimension (3D) culture system. Through utilizing 3D dynamic culture, this protocol successfully obtained large-scale exosomes from SF-MSC culture supernatants. Exosomes were harvested by ultracentrifugation and verified by a transmission electron microscope, nanoparticle transmission assay, and western blotting. Also, the microbiological safety of exosomes was detected. Results of exosome detection suggest that this approach can produce a large number of good manufacturing practices (GMP)-grade exosomes. These exosomes could be utilized in exosome biology research and clinical osteoarthritis treatment.

Here, we present a protocol to produce a large number of GMP-grade exosomes from synovial fluid mesenchymal stem cells using a 3D bioreactor.

3D生物反应器培养大规模制备滑液间充质干细胞来源外泌体

Exosomes secreted by mesenchymal stem cells (MSCs) have been suggested as promising candidates for cartilage injuries and osteoarthritis treatment. ...

恶性疟原虫 通过人工膜喂养进行配子体培养和蚊子感染

恶性疟原虫通过人工膜喂养进行配子体培养和蚊子感染