作者想感谢约翰弗雷和皮特森本蚊虫饲养。我们也感谢恭戴维斯和加里Radice与血细胞显微镜的帮助。这项研究是由美国里士满大学艺术和科学的夏季奖学金AA Qayum和教师授予答：特朗。

1。在血细胞收集提前准备<ol><li>准备一个血淋巴稀释剂的解决方案，施耐德公司的60％中等，10％胎牛血清（FBS），和30％的柠檬酸缓冲由（抗凝血剂; 98毫米氢氧化钠，186 mM的氯化钠，1.7 mM的EDTA，41 mM的柠檬酸酸，pH值4.5） 11。设为1.0毫升分装，可在-20 ° C存储只有一次使用每个等份（见材料）。 </li><li>准备通过计分的一端1厘米直径的圆，用玻璃蚀刻工具的玻璃显微镜载玻片（75 × 25 × 1毫米）。另外，有两个预蚀刻界的显微镜幻灯片就可以买到（如费舍尔科学或EMS来源）。蚊子的每一个玻片上，将是必要的。 </li><li>准备一个玻璃针（见材料）拉到按照以下建议设置（萨特仪器，模型P - 87）： <table border="1"><tbody><tr><td>热斜坡5 </td></tr><tr><td>拉：45 </td></tr><tr><td> VEL：75 </td></tr><tr><td>时间：175 </td></tr><tr><td>压力580 </td></tr></tbody></table> 使用本拉马建议的设置，我们构建柄长度约3毫米的总长度约500毫米的针头。预拉的针头也可购买（如Tritech研究）。 </li></ol> 2。提前血细胞收集在微量制备<ol><li>根据制造商的指示，拉针插入微量持针器一个。回填微量（见材料）管，注射器和矿物油拉玻璃针根据制造商的指示。 </li></ol> 3。雌性蚊子在血细胞收集前的准备<ol><li> 15个成年女性吸进小实体保持架可以在与冰接触的容器。浸入深度不够冰蚊子的笼子是使整个笼和各方的高度与冰的接触。这将确保所有的蚊子将被暴露在寒冷。 冷麻醉蚊子约8分钟或直到他们不再移动。 </li><li>放置到一个抑郁症的幻灯片一个蚊子。这张幻灯片将作为注射部位。 </li></ol> 4。血细胞集合<ol><li>设置微量采取拉玻璃针12μL的血淋巴稀释剂注入蚊子之间的第七和第八腹节的解决方案。 3-3.5总占用量12μL液，可以注入约3蚊子。蚊子应用镊子轻轻地举行，而注射执行到位。针尖应维持在一个小体积的稀释剂，以防止被注入矿物油。注射后的腹部应该看看“填充”或臃肿。 </li><li>地方在一个单独的干净的容器中注入蚊子在冰上5分钟恢复。孵化在蚊子体内的抗凝血剂的稀释剂的解决方案将帮助打跑坚持内部组织的血细胞。平均3至5新鲜蚊子可以注射期间每5分钟的恢复时期。 </li><li>经过5分钟的恢复，剪断过腿，翅膀，和每个使用微型剪刀蚊子的腹部（第八部分）的一角。这项工作应在恢复杯置于冰上。切断腿和翅膀，降低被转移到收集幻灯片尺度的可能性。注：不砍腿和翅膀太接近胸附着点，或你的风险创造了血淋巴逃脱其他开口。淋巴只应收集通过在腹部末端创建开幕。 </li><li>与70％的乙醇玻璃蚀刻显微镜幻灯片洗净，并用kimwipe。位置之一注入和回收蚀刻圈的外缘的幻灯片上的蚊子。设置微量拿起12新鲜的血淋巴稀释剂液，注入蚊子在其mesothorax侧方音量。提供最，但不是整个卷稀释剂（约8 - 10μL），以防止被注入矿物油。针尖应维持在一个小体积的稀释剂。 </li><li>蚊子，因此，削减腹部开放1厘米的圆的边缘位置。注射后稀释血淋巴应收集标记的区域内。一旦针头从mesothoracic注射部位拆除，恢复举行蚊子两钳之间的武器，并与这些镊子轻轻挤压腹部，直接上蚀刻的圆面积的血淋巴。 </li><li>允许稀释血淋巴干幻灯片，约10分钟，或直至它是明显的干。 </li></ol> 5。血细胞固定和染色<ol><li>染色每张幻灯片分别HEMA3 7（见材料）：DIP小号立德在固定液5次为1秒每次，在我的解决方案，最后浸在溶液II的幻灯片各1秒3次;各1秒的3倍Dipslide。 DI水的幻灯片冲洗背面。前面的幻灯片不应该冲洗。吸干干蚀刻圈之外，含有染色组织的幻灯片的前面。 </li><li>围绕蚀刻中心申请安装介质（如书珥/摩或Polymount），并在其上​​的盖玻片。按到盖玻片（25 × 25毫米）轻微，所以，安装介质扩散到整个区域覆盖盖玻片（含染色组织包括蚀刻圈）。允许幻灯片干燥至少3小时或过夜。血细胞应被视为2个星期内，以下为最佳的可视化和成像收集。 </li></ol> 6。代表性的成果图1A - C的收集从埃及伊蚊成年女性的固定和HEMA3染色的血细胞的例子所示。图1A显示了我们作为基于它的体积小，球形球状的形状和高核浆比例（550X的放大倍率）11一个prohemocyte确定的血细胞。图1b显示了其椭球的形状和高细胞质核比（550X的放大倍率）为基础的oenocytoid分类血细胞。最后，图1C显示一个粒型血细胞（550X的放大倍率）。粒颗粒性质，而且往往在形状和行为更变形虫，因为他们坚持以玻璃表面。使用原先公布的血细胞打字和恢复11的标准，我们的收集方法取得了类似的总血细胞数量和我们同样发现，粒细胞构成的总血细胞的比例最大的11（图2）。 <img src="/files/ftp_upload/2772/2772fig1.jpg" alt="图1" /> 图1。一个有代表性的HEMA3固定，染色prohemocyte光显微镜图像（A）oenocytoid（B）和粒（三）从AE。埃及伊蚊成年女性。 C =细胞质，N ​​=核摹=颗粒。 <img src="/files/ftp_upload/2772/2772fig2.jpg" alt="图2"/> 图2。总计数± SE的每雌蚊获得所有的血细胞和粒细胞，我们的血淋巴中的稀释和收集方法。

<ol>
	<li>Enayati, A., Hemingway, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Malaria+management:+past,+present,+and+future.">Malaria management: past, present, and future.</a> Annu. Rev. Entomol. 55, 569-569 (2010).</li><li>Medlock, J., Luz, P. M., Struchiner, C. J., Galvani, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+transgenic+mosquitoes+on+dengue+virulence+to+humans+and+mosquitoes.">The impact of transgenic mosquitoes on dengue virulence to humans and mosquitoes.</a> The American Naturalist. 174, 565-565 (2009).</li><li>Baton, L. A., Garver, L., Xi, Z., 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=Functional+genomics+studies+on+the+innate+immunity+of+disease+vectors.">Functional genomics studies on the innate immunity of disease vectors.</a> Insect Sci. 15, 15-15 (2008).</li><li>Thomas, R. E., Wu, W. K., Verleye, D., Rai, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Midgut+basal+lamina+thickness+and+dengue-1+virus+dissemination+rates+in+laboratory+strains+of+Aedes+albopictus+(Diptera:+Culicidae).">Midgut basal lamina thickness and dengue-1 virus dissemination rates in laboratory strains of Aedes albopictus (Diptera: Culicidae).</a> J. Med. Entomol. 30, 326-326 (1993).</li><li>Bartholomay, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profiling+infection+responses+in+the+haemocytes+of+the+mosquito,+Aedes+aegypti.">Profiling infection responses in the haemocytes of the mosquito, Aedes aegypti.</a> Insect Mol. Biol. 16, 761-761 (2007).</li><li>Strand, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+insect+cellular+immune+response.">The insect cellular immune response.</a> Insect Sci. 15, 1-1 (2008).</li><li>Hillyer, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-associated+mortality+in+immune+challenged+mosquitoes+(Aedes+aegypti)+correlates+with+a+decrease+in+haemocyte+numbers.">Age-associated mortality in immune challenged mosquitoes (Aedes aegypti) correlates with a decrease in haemocyte numbers.</a> Cell. Microbiol. 7, 39-39 (2005).</li><li>Chen, C. C., Laurence, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+study+on+humoral+encapsulation+of+microfilariae:+establishment+of+technique+and+description+of+reaction.">In vitro study on humoral encapsulation of microfilariae: establishment of technique and description of reaction.</a> International Journal for Parasitology. 17, 781-781 (1987).</li><li>Beerntsen, B. T., Christensen, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dirofilaria+immitis:+effects+on+hemolymph+polypeptide+synthesis+in+Aedes+aegypti+during+melanotic+encapsulation+reactions+against+Microfilariae.">Dirofilaria immitis: effects on hemolymph polypeptide synthesis in Aedes aegypti during melanotic encapsulation reactions against Microfilariae.</a> Exp. Parasitol. 71, 406-406 (1990).</li><li>Hillyer, J. F., Schmidt, S. L., Christensen, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+antibacterial+innate+immune+response+by+the+mosquito+Aedes+aegypti+is+mediated+by+hemocytes+and+independent+of+Gram+type+and+pathogenicity.">The antibacterial innate immune response by the mosquito Aedes aegypti is mediated by hemocytes and independent of Gram type and pathogenicity.</a> Microb. Infect. 6, 448-448 (2004).</li><li>Castillo, J. C., Robertson, A. E., Strand, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+hemocytes+from+the+mosquitoes+Anopheles+gambiae+and+Aedes+aegypti.">Characterization of hemocytes from the mosquitoes Anopheles gambiae and Aedes aegypti.</a> Insect Biochem. Mol. Biol. 36, 891-891 (2006).</li></ol>

这里描述的血细胞的采集方法是从先前公布的方法修改，并允许一个孤立伊蚊血细胞的清洁准备用更少的步骤。虽然我们特别感兴趣的特征伊蚊血细胞种群的谎言，我们相信这种技术可以应用于其他蚊虫群体后进行的初步试验，以确定正确的注入量。本协议代替盐溶液中使用抗凝血剂的缓冲区，并产生血细胞的人口，准确地反映体内的数字。然而，我们还没有确定是否适合我?...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td>施耐德公司的培养基</td><td> Sigma - Aldrich公司</td><td> S0146 </td><td></td></tr><tr><td>胎牛血清</td><td> Sigma - Aldrich公司</td><td> F0643 </td><td></td></tr><tr><td> Hema3染色套件</td><td>费舍尔</td><td> 123-869 </td><td></td></tr><tr><td>玻璃针 （高硼硅与长丝） </td><td>萨特仪器</td><td> BF100 - 78 - 10 </td><td> 1.0毫米OD和0.78毫米ID </td></tr><tr><td>针拉马</td><td>萨特仪器</td><td> P - 87型</td><td></td></tr><tr><td>微量</td><td> Tritech研究</td><td> MINJ - PD </td><td></td></tr><tr><td>持针器</td><td> Tritech研究</td><td> MINJ - 4 </td><td></td></tr></tbody></table>

a protocol for collecting and staining hemocytes from the yellow fever mosquito aedes aegypti

蚊子是一些引起疾病的病原体，如黄热病病毒，疟原虫和丝虫的载体。实验室正在调查抗病原体的先天免疫系统产生，耐火材料等病原体1，2的转基因蚊子的希望病媒物种的组成部分。蚊子先天免疫系统由几行防御3。设法逃脱上皮内衬的蚊子中肠 4施加的障碍的病原体进入血淋巴中遇到的循环血细胞，重要的细胞成分，封装和吞噬病原体 5 ，6。研究人员还没有发现蚊子造血组织的证据，现有的证据表明，血细胞数量是固定的成虫羽化，实际数字可能下降，因为蚊子的7岁。妥善收集，并确定从医学上重要的昆虫的血细胞的能力，细胞免疫功能的研究是一个必不可少的步骤。但是，蚊子的小规模和数量有限的血淋巴构成收集的免疫细胞所面临的挑战。 两个建立的方法，包括收集蚊子血细胞血淋巴，从切割长鼻8，驱逐和位移量（灌注），其中生理盐水注入膜necklike地区之间的头部和胸部（即子宫颈癌）和灌注血淋巴收集从远端区域的腹部9，10撕裂开放。然而，这些技术是有限的，低血细胞恢复和由脂肪体细胞，分别为 11可能造成的污染。最近提到的方法使用抗凝血剂的缓冲区，以高喷射/恢复提高免疫细胞的恢复，同时减少污染规模和内部组织11水平。虽然这种方法允许收集和维持血细胞原代培养方法的改进，它需要注入和收集的步骤是没有必要的，如果下游的目标是收集，固定和染色诊断血细胞的数量。在这里，我们展示我们收集蚊子血淋巴的方法相结合的简单灌注，高喷射技术的准确性伊蚊血细胞分离干净准备，代替盐溶液中使用抗凝血剂的缓冲区。

Watch this Scientific Journal Video about A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti at JoVE.com

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

一种简捷而准确的方法来收集和染色描述的蚊子血细胞。我们的方法结合灌注简单，精度高喷射技术伊蚊血细胞分离干净准备。这种方法有利于血细胞类型的研究需要的知识和它的丰富。

从黄热病的蚊子的血细胞的收集和染色的议定书埃及伊蚊</em

 Mosquitoes are vectors for a number  of disease-causing pathogens such as the yellow fever virus, malaria parasites  and filarial worms. Laboratories are ...

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Research

JoVE Journal

Immunology and Infection

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

15.2K 次观看.  University of Richmond. 一种简捷而准确的方法来收集和染色描述的蚊子血细胞。我们的方法结合灌注简单，精度高喷射技术伊蚊血细胞分离干净准备。这种方法有利于血细胞类型的研究需要的知识和它的丰富。

视频: A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

Protocol for Production of a Genetic Cross of the Rodent Malaria Parasites

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to successful identification of genes or loci underlying various traits in malaria parasites of rodents1-3 and humans4-6. The malaria parasite Plasmodium yoelii is one of many malaria species isolated from wild African rodents and has been adapted to grow in laboratories. This species reproduces many of the biologic characteristics of the human malaria parasites; genetic markers such as microsatellite and amplified fragment length polymorphism (AFLP) markers have also been developed for the parasite7-9. Thus, genetic studies in rodent malaria parasites can be performed to complement research on Plasmodium falciparum. Here, we demonstrate the techniques for producing a genetic cross in P. yoelii that were first pioneered by Drs. David Walliker, Richard Carter, and colleagues at the University of Edinburgh10.

Genetic crosses in P. yoelii and other rodent malaria parasites are conducted by infecting mice Mus musculus with an inoculum containing gametocytes of two genetically distinct clones that differ in phenotypes of interest and by allowing mosquitoes to feed on the infected mice 4 days after infection. The presence of male and female gametocytes in the mouse blood is microscopically confirmed before feeding. Within 48 hrs after feeding, in the midgut of the mosquito, the haploid gametocytes differentiate into male and female gametes, fertilize, and form a diploid zygote (Fig. 1). During development of a zygote into an ookinete, meiosis appears to occur11. If the zygote is derived through cross-fertilization between gametes of the two genetically distinct parasites, genetic exchanges (chromosomal reassortment and cross-overs between the non-sister chromatids of a pair of homologous chromosomes; Fig. 2) may occur, resulting in recombination of genetic material at homologous loci. Each zygote undergoes two successive nuclear divisions, leading to four haploid nuclei. An ookinete further develops into an oocyst. Once the oocyst matures, thousands of sporozoites (the progeny of the cross) are formed and released into mosquito hemoceal. Sporozoites are harvested from the salivary glands and injected into a new murine host, where pre-erythrocytic and erythrocytic stage development takes place. Erythrocytic forms are cloned and classified with regard to the characters distinguishing the parental lines prior to genetic linkage mapping. Control infections of individual parental clones are performed in the same way as the production of a genetic cross.

Genetic crosses of rodent malaria parasites are performed by feeding two genetically distinct parasites to mosquitoes. Recombinant progeny are cloned from mouse blood after allowing mosquitoes to bite infected mice. This video shows how to produce genetic crosses of Plasmodium yoelii and is applicable to other rodent malaria parasites.

生产的灭鼠疟疾寄生虫的遗传交叉议定书“

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to ...

科研

免疫与感染

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito (Aedes aegypti) and Tick (Rhipicephalus microplus) G Protein-coupled Receptors

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes. The majority of them belong to the superfamily of G protein-coupled receptors (GPCRs). We have focused on characterizing arthropod kinin receptors from the tick and mosquito. Arthropod kinins are multifunctional neuropeptides with myotropic, diuretic, and neurotransmitter function. Here, a method for systematic analyses of structure-activity relationships of insect kinins on two heterologous kinin receptor-expressing systems is described. We provide important information relevant to the development of biostable kinin analogs with the potential to disrupt the diuretic, myotropic, and/or digestive processes in ticks and mosquitoes.
The kinin receptors from the southern cattle tick, Boophilus microplus (Canestrini), and the mosquito Aedes aegypti (Linnaeus), were stably expressed in the mammalian cell line CHO-K1. Functional analyses of these receptors were completed using a calcium bioluminescence plate assay that measures intracellular bioluminescence to determine cytoplasmic calcium levels upon peptide application to these recombinant cells. This method takes advantage of the aequorin protein, a photoprotein isolated from luminescent jellyfish. We transiently transfected the aequorin plasmid (mtAEQ/pcDNA1) in cell lines that stably expressed the kinin receptors. These cells were then treated with the cofactor coelenterazine, which complexes with intracellular aequorin. This bond breaks in the presence of calcium, emitting luminescence levels indicative of the calcium concentration. As the kinin receptor signals through the release of intracellular calcium, the intensity of the signal is related to the potency of the peptide.
This protocol is a synthesis of several previously described protocols with modifications; it presents step-by-step instructions for the stable expression of GPCRs in a mammalian cell line through functional plate assays (Staubly et al., 2002 and Stables et al., 1997). Using this methodology, we were able to establish stable cell lines expressing the mosquito and the tick kinin receptors, compare the potency of three mosquito kinins, identify critical amino acid positions for the ligand-receptor interaction, and perform semi-throughput screening of a peptide library. Because insect kinins are susceptible to fast enzymatic degradation by endogenous peptidases, they are severely limited in use as tools for pest control or endocrinological studies. Therefore, we also tested kinin analogs containing amino isobutyric acid (Aib) to enhance their potency and biostability. This peptidase-resistant analog represents an important lead in the development of biostable insect kinin analogs and may aid in the development of neuropeptide-based arthropod control strategies.

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito Aedes aegypti and Tick Rhipicephalus microplus G Protein-coupled Receptors

This protocol provides instructions for clonal-cell line selection and a calcium bioluminescence assay to analyze the structure-activity relationships of synthesized arthropod neuropeptides on their cognate GPCRs. This assay can be used for receptor deorphanization and structure-activity relationship studies for synthetic analog design and peptide/drug-lead discovery.

一个钙生物发光检测蚊虫的功能分析（埃及伊蚊）和刻度（镰形扇头microplus）G蛋白偶联受体

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes.  The ...

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 ...

A Protocol for Analyzing Hepatitis C Virus Replication

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HCV is an enveloped RNA virus belonging to the family Flaviviridae. Current treatment is not fully effective and causes adverse side effects. There is no HCV vaccine available. Thus, continued effort is required for developing a vaccine and better therapy. An HCV cell culture system is critical for studying various stages of HCV growth including viral entry, genome replication, packaging, and egress. In the current procedure presented, we used a wild-type intragenotype 2a chimeric virus, FNX-HCV, and a recombinant FNX-Rluc virus carrying a Renilla luciferase reporter gene to study the virus replication. A human hepatoma cell line (Huh-7 based) was used for transfection of in vitro transcribed HCV genomic RNAs. Cell-free culture supernatants, protein lysates and total RNA were harvested at various time points post-transfection to assess HCV growth. HCV genome replication status was evaluated by quantitative RT-PCR and visualizing the presence of HCV double-stranded RNA. The HCV protein expression was verified by Western blot and immunofluorescence assays using antibodies specific for HCV NS3 and NS5A proteins. HCV RNA transfected cells released infectious particles into culture supernatant and the viral titer was measured. Luciferase assays were utilized to assess the replication level and infectivity of reporter HCV. In conclusion, we present various virological assays for characterizing different stages of the HCV replication cycle.

Hepatitis C Virus (HCV) is a major human pathogen that causes liver disorders, including cirrhosis and cancer. An HCV infectious cell culture system is essential for understanding the molecular mechanism of HCV replication and developing new therapeutic approaches. Here we describe a protocol to investigate various stages of the HCV replication cycle.

一个协议，用于分析丙型肝炎病毒复制

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and ...

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the goal of this protocol is to develop a method to visualize the pathogen invasion in a specific immune compartment of flies, namely hemocytes. Using the method presented here, up to 3 &#215; 106 live hemocytes can be obtained from 200 Drosophila 3rd instar larvae in 30 min for ex vivo infection. Alternatively, hemocytes can be infected in vivo through injection of 3rd instar larvae followed by hemocyte extraction up to 24 h post-infection. These infected primary cells were fixed, stained, and imaged using confocal microscopy. Then, 3D representations were generated from the images to definitively show pathogen invasion. Additionally, high-quality RNA for qRT-PCR can be obtained for the detection of pathogen mRNA following infection, and sufficient protein can be extracted from these cells for Western blot analysis. Taken together, we present a method for definite reconciliation of pathogen invasion and confirmation of infection using bacterial and viral pathogen types and an efficient method for hemocyte extraction to obtain enough live hemocytes from Drosophila larvae for ex vivo and in vivo infection experiments.

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

This method demonstrates how to visualize pathogen invasion into insect cells with three-dimensional (3D) models. Hemocytes from Drosophila larvae were infected with viral or bacterial pathogens, either ex vivo or in vivo. Infected hemocytes were then fixed and stained for imaging with a confocal microscope and subsequent 3D cellular reconstruction.

从果蝇幼虫中提取包囊微生物感染及分析

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the ...

Co-staining Blood Vessels and Nerve Fibers in Adipose Tissue

Recent studies have highlighted the critical role of angiogenesis and sympathetic innervation in adipose tissue remodeling during the development of obesity. Therefore, developing an easy and efficient method to document the dynamic changes in adipose tissue is necessary. Here, we describe a modified immunofluorescent approach that efficiently co-stains blood vessels and nerve fibers in adipose tissues. Compared to traditional and recently developed methods, our approach is relatively easy to follow and more efficient in labeling the blood vessels and nerve fibers with higher densities and less background. Moreover, the higher resolution of the images further allows us to accurately measure the area of the vessels, amount of branching, and length of the fibers by open source software. As a demonstration using our method, we show that brown adipose tissue (BAT) contains higher amounts of blood vessels and nerve fibers compared to white adipose tissue (WAT). We further find that among the WATs, subcutaneous WAT (sWAT) has more blood vessels and nerve fibers compared to epididymal WAT (eWAT). Our method thus provides a useful tool for investigating adipose tissue remodeling.

New blood vessel formation and sympathetic innervation play pivotal roles in adipose tissue remodeling. However, there remain technical issues in visualizing and quantitatively measuring adipose tissue. Here we present a protocol to successfully label and quantitatively compare the densities of blood vessels and nerve fibers in different adipose tissues.

脂肪组织中的血管和神经纤维的共染色

Recent studies have highlighted the critical role of angiogenesis and sympathetic innervation in adipose tissue remodeling during the development of ...

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy for the Replacement, Reduction, and Refinement of the laboratory animal testing. The development of in vitro approaches is recommended at the WHO and European levels as alternatives to the NIH test for evaluating the rabies vaccine potency. At the surface of the rabies virus (RABV) particle, trimers of glycoprotein constitute the major immunogen to induce Viral Neutralizing Antibodies (VNAbs). An ELISA test, where Neutralizing Monoclonal Antibodies (mAb-D1) recognize the trimeric form of the glycoprotein, has been developed to determine the contents of the native folded trimeric glycoprotein along with the production of the vaccine batches. This in vitro potency test demonstrated a good concordance with the NIH test and has been found suitable in collaborative trials by RABV vaccine manufacturers and OMCLs. Avoidance of animal use is an achievable objective in the near future.
The method presented is based on an indirect ELISA sandwich immunocapture using the mAb-D1 which recognizes the antigenic sites III (aa 330 to 338) of the trimeric RABV glycoprotein, i.e., the immunogenic RABV antigen. mAb-D1 is used for both coating and detection of glycoprotein trimers present in the vaccine batch. Since the epitope is recognized because of its conformational properties, the potentially denatured glycoprotein (less immunogenic) cannot be captured and detected by the mAb-D1. The vaccine to be tested is incubated in a plate sensitized with the mAb-D1. Bound trimeric RABV glycoproteins are identified by adding the mAb-D1 again, labeled with peroxidase and then revealed in the presence of substrate and chromogen. Comparison of the absorbance measured for the tested vaccine and the reference vaccine allows for the determination of the immunogenic glycoprotein content.

Here we describe an indirect ELISA sandwich immunocapture to determine the immunogenic glycoprotein contents in rabies vaccines. This test uses a neutralizing Monoclonal Antibody (mAb-D1) recognizing glycoprotein trimers. It is an alternative to the in vivo NIH test to follow the consistency of vaccine potency during production.

体外ELISA测试评估狂犬病疫苗效力

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy ...

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine the rate of infection, disseminated infection, and potential transmission of the virus in the mosquito population in question. These procedures are widely utilized with various modifications in vector competence evaluations globally. They are important in determining the potential role that a given mosquito (i.e., species, population, individual) may play in the transmission of a given agent.

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

The presented protocol can determine the vector competence of Aedes aegypti mosquito populations for a given virus, such as Zika, in a containment setting.

使用寨卡病毒对 埃及伊 蚊的载体能力分析

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine ...

Propagation of the Microsporidian Parasite Edhazardia aedis in Aedes aegypti Mosquitoes

Edhazardia aedis is a microsporidian parasite of Aedes aegypti mosquitoes, a disease vector that transmits multiple arboviruses which cause millions of disease cases each year. E. aedis causes mortality and reduced reproductive fitness in the mosquito vector and has been explored for its potential as a biocontrol agent. The protocol we present for culturing E. aedis is based on its natural infection cycle, which involves both horizontal and vertical transmission at different life stages of the mosquito host. Ae. aegypti mosquitoes are exposed to spores in the larval stage. These infected larvae then mature into adults and transmit the parasite vertically to their offspring. Infected offspring are then used as a source of spores for future horizontal transmission. Culturing E. aedis can be challenging to the uninitiated given the complexities of the parasite&#8217;s life cycle, and this protocol provides detailed guidance and visual aids for clarification.

Propagation of the Microsporidian Parasite Edhazardia aedis in Aedes aegypti Mosquitoes

A protocol to culture the microsporidian parasite Edhazardia aedis. The parasite is passaged from one generation of Aedes aegypti mosquitoes to the next via horizontal transfer at the larval stage followed by vertical transmission at the adult stage. Live sporoplasms survive long-term in infected eggs.

在伊蚊中传播微孢子寄生虫Edhazardia aedis

Edhazardia aedis is a microsporidian parasite of Aedes aegypti mosquitoes, a disease vector that transmits multiple arboviruses which cause millions of ...

Feeding and Quantifying Animal-Derived Blood and Artificial Meals in Aedes aegypti Mosquitoes

Females of certain mosquito species can spread diseases while biting vertebrate hosts to obtain protein-rich blood meals required for egg development. In the laboratory, researchers can deliver animal-derived and artificial blood meals to mosquitoes via membrane feeders, which allow for manipulation of meal composition. Here, we present methods for feeding blood and artificial blood meals to Aedes aegypti mosquitoes and quantifying the volume consumed by individual females.
Targeted feeding and quantification of artificial/blood meals have broad uses, including testing the effects of meal components on mosquito behavior and physiology, delivering pharmacological compounds without injection, and infecting mosquitoes with specific pathogens. Adding fluorescein dye to the meal prior to feeding allows for subsequent meal size quantification. The meal volume consumed by mosquitoes can be measured either by weight, if the females are to be used later for behavioral experiments, or by homogenizing individual females in 96-well plates and measuring fluorescence levels using a plate reader as an endpoint assay. Meal size quantification can be used to determine whether changing the meal components alters the meal volume ingested or if meal consumption differs between mosquito strains. Precise meal size quantification is also critical for downstream assays, such as those measuring effects on host attraction or fecundity. The methods presented here can be further adapted to track meal digestion over the course of days or to include multiple distinguishable markers added to different meals (like nectar and blood) to quantify the consumption of each meal by a single mosquito.
These methods allow researchers to singlehandedly perform high-throughput measurements to compare the meal volume consumed by hundreds of individual mosquitoes. These tools will therefore be broadly useful to the community of mosquito researchers for answering diverse biological questions.

Feeding and Quantifying Animal-Derived Blood and Artificial Meals in Aedes aegypti Mosquitoes

The goal of this protocol is to deliver animal-derived and artificial blood meals to Aedes aegypti mosquitoes through an artificial membrane feeder and precisely quantify the volume of meal ingested.

在 伊 蚊中喂养和量化动物衍生的血液和人工膳食

Females of certain mosquito species can spread diseases while biting vertebrate hosts to obtain protein-rich blood meals required for egg development. In ...