Name: a protocol for collecting and staining hemocytes from the yellow fever mosquito aedes aegypti
Uploaded: 2011-05-16T12:00:00
Description: Watch this Scientific Journal Video about A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti at JoVE.com

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...

<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 ...

Research

JoVE Journal

Immunology and Infection

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

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

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

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.

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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 ...

Utilization of Capsules for Negative Staining of Viral Samples within Biocontainment

利用胶囊对生物达标中病毒样品的阴性染色

Transmission electron microscopy (TEM) is used to observe the ultrastructure of viruses and other microbial pathogens with nanometer resolution. Most ...

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

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

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 ...

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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

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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

Propagation of the Microsporidian Parasite Edhazardia aedis in Aedes aegypti Mosquitoes

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

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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 ...