Name: tick artificial membrane feeding for ixodes scapularis
Uploaded: 2022-11-30T14:00:00
Description: Watch this Scientific Journal Video about Tick Artificial Membrane Feeding for Ixodes scapularis at JoVE.com

1. 准备蜱膜室
<ol><li>用70%乙醇擦拭，准备一个平坦的无孔表面，例如玻璃平面或陶瓷涂层的金属底座，然后用单层保鲜膜覆盖，确保保鲜膜平坦，没有气泡或皱纹（见 图1A）。</li>
	<li>将100%人造丝镜头清洁纸胶带到准备好的表面上。确保它平坦并用胶带在纸张的所有四个面上略微拉紧。可以使用步骤 1.3 中描述的体积将两张 4 英寸 x 6 英寸的透镜纸制成膜。 ?...

<ol>
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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inability+of+a+variant+strain+of+Anaplasma+phagocytophilum+to+infect+mice.">Inability of a variant strain of Anaplasma phagocytophilum to infect mice.</a> The Journal of Infectious Diseases. 188 (11), 1757-1763 (2003).</li><li>Koci, J., Bernard, Q., Yang, X., Pal, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Borrelia+burgdorferi+surface+protein+Lmp1+facilitates+pathogen+dissemination+through+ticks+as+studied+by+an+artificial+membrane+feeding+system.">Borrelia burgdorferi surface protein Lmp1 facilitates pathogen dissemination through ticks as studied by an artificial membrane feeding system.</a> Scientific Reports. 8 (1), 1910 (2018).</li><li>Levin, M. L., Schumacher, L. 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=Manual+for+maintenance+of+multi-host+ixodid+ticks+in+the+laboratory.">Manual for maintenance of multi-host ixodid ticks in the laboratory.</a> Experimental and Applied Acarology. 70 (3), 343-367 (2016).</li><li>Hart, T., Yang, X., Pal, U., Lin, Y. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Lyme+borreliae+proteins+promoting+vertebrate+host+blood-specific+spirochete+survival+in+Ixodes+scapularis+nymphs+using+artificial+feeding+chambers.">Identification of Lyme borreliae proteins promoting vertebrate host blood-specific spirochete survival in Ixodes scapularis nymphs using artificial feeding chambers.</a> Ticks and Tick-Borne Diseases. 9 (5), 1057-1063 (2018).</li><li>Hart, T. M., 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=Host+tropism+determination+by+convergent+evolution+of+immunological+evasion+in+the+Lyme+disease+system.">Host tropism determination by convergent evolution of immunological evasion in the Lyme disease system.</a> PLoS Pathogens. 17 (7), (2021).</li><li>Bernard, Q., 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=Plasticity+in+early+immune+evasion+strategies+of+a+bacterial+pathogen.">Plasticity in early immune evasion strategies of a bacterial pathogen.</a> Proceedings of the National Academy of Sciences. 115 (16), 3788-3797 (2018).</li><li>Pierce, A. E., Pierce, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+note+on+the+cultivation+of+Boophilus+microplus+(Canestrini,+1887)+(Ixodidae:+Acarina)+on+the+embyonated+hen+egg.">A note on the cultivation of Boophilus microplus (Canestrini, 1887) (Ixodidae: Acarina) on the embyonated hen egg.</a> Australian Veterinary Journal. 32 (6), 144-146 (1956).</li><li>Doube, B. M., Kemp, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+temperature,+relative+humidity+and+host+factors+on+the+attachment+and+survival+of+Boophilus+microplus+(Canestrini)+larvae+to+skin+slices.">The influence of temperature, relative humidity and host factors on the attachment and survival of Boophilus microplus (Canestrini) larvae to skin slices.</a> International Journal for Parasitology. 9 (5), 449-454 (1979).</li><li>Kr&#246;ber, T., Guerin, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+feeding+assay+to+test+acaricides+for+control+of+hard+ticks.">An in vitro feeding assay to test acaricides for control of hard ticks.</a> Pest Management Science. 63 (1), 17-22 (2007).</li><li>Kuhnert, F., Diehl, P. A., Guerin, P. 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+life-cycle+of+the+bont+tick+Amblyomma+hebraeum+in+vitro.">The life-cycle of the bont tick Amblyomma hebraeum in vitro.</a> International Journal for Parasitology. 25 (8), 887-896 (1995).</li><li>Kr&#246;ber, T., Guerin, P. M. <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+feeding+assays+for+hard+ticks.">In vitro feeding assays for hard ticks.</a> Trends in Parasitology. 23 (9), 445-449 (2007).</li><li>Andrade, J. J., Xu, G., Rich, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+silicone+membrane+for+in+vitro+feeding+of+Ixodes+scapularis+(Ixodida:+Ixodidae).">A silicone membrane for in vitro feeding of Ixodes scapularis (Ixodida: Ixodidae).</a> Journal of Medical Entomology. 51 (4), 878-879 (2014).</li><li>Oliver, J. 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=Infection+of+immature+Ixodes+scapularis+(Acari:+Ixodidae)+by+membrane+feeding.">Infection of immature Ixodes scapularis (Acari: Ixodidae) by membrane feeding.</a> Journal of Medical Entomology. 53 (2), 409-415 (2016).</li><li>Oliver, J. 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=Growth+dynamics+and+antibiotic+elimination+of+symbiotic+Rickettsia+buchneri+in+the+tick+Ixodes+scapularis+(Acari:+Ixodidae).">Growth dynamics and antibiotic elimination of symbiotic Rickettsia buchneri in the tick Ixodes scapularis (Acari: Ixodidae).</a> Applied and Environmental Microbiology. 87 (3), (2021).</li><li>Graham, E. E., Poland, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+Fluon+conditioning+for+capturing+cerambycid+beetles+in+different+trap+designs+and+persistence+on+panel+traps+over+time.">Efficacy of Fluon conditioning for capturing cerambycid beetles in different trap designs and persistence on panel traps over time.</a> Journal of Economic Entomology. 105 (2), 395-401 (2012).</li><li>Munderloh, U. G., Liu, Y., Wang, M., Chen, C., Kurtti, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment,+maintenance+and+description+of+cell+lines+from+the+tick+Ixodes+scapularis.">Establishment, maintenance and description of cell lines from the tick Ixodes scapularis.</a> Journal of Parasitology. 80 (4), 533-543 (1994).</li><li>Lehane, 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=Prevalence+of+single+and+coinfections+of+human+pathogens+in+Ixodes+ticks+from+five+geographical+regions+in+the+United+States,+2013-2019.">Prevalence of single and coinfections of human pathogens in Ixodes ticks from five geographical regions in the United States, 2013-2019.</a> Ticks and Tick-Borne Diseases. 12 (2), (2021).</li><li>Gonz&#225;lez, J., Bickerton, M., Toledo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+artificial+membrane+feeding+for+ixodid+ticks.">Applications of artificial membrane feeding for ixodid ticks.</a> Acta Tropica. 215, (2021).</li><li>Kr&#243;l, N., 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=Evaluating+transmission+paths+for+three+different+Bartonella+spp.+in+Ixodes+ricinus+ticks+using+artificial+feeding.">Evaluating transmission paths for three different Bartonella spp. in Ixodes ricinus ticks using artificial feeding.</a> Microorganisms. 9 (5), 901 (2021).</li><li>Anderson, J. F., Magnarelli, L. A. <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+ticks.">Biology of ticks.</a> Infectious Disease Clinics of North America. 22 (2), 195-215 (2008).</li></ol>

蜱虫的人工膜喂养为各种实验程序提供了有用的工具，但不太可能取代所有应用的动物喂养。在没有动物喂养的情况下，在所有生命阶段维持大量蜱虫通常是站不住脚的。相反，人工饲喂系统对于其他目的很有价值，例如用模型宿主不支持的病原体感染蜱虫，在简化的摄食环境中评估化合物或微生物的受控剂量对蜱虫的影响，或饲养依赖实验室中不易获得的宿主动物的小蜱虫菌落8</s...

蜱传疾病严重影响着全世界人类和动物的健康，2004年至2016年占美国所有病媒相关疾病的三分之二以上1。此外，近年来病例数一直在增长，越来越多的人和牲畜受到蜱虫及其相关疾病的影响2，3。虽然病例数上升趋势的原因可能有很多，但气候变化是一个重要因素3，4.预计蜱传疾病病例数量的持续增加突出表明需要开发新工具来调查蜱与其传播的病原体之间的关系。
众所周知，蜱在进食过程中会经历生理和基因表达的变化，这些变化在病原体传播中起作用5，6。使用动物模型进行研究检查全部和部分摄食对病原体传播和获取的影响可能很困难，特别是在啮齿动物模型不易被特定病原体感染的情况下。例如， 无形体吞噬细胞变体 -1菌株在 肩突硬蜱 和鹿之间自然传播，但无法感染小鼠，使实验室中的蜱感染复杂化7。人工喂养系统也可以通过使用具有抑制传播或感染的基因缺失的转基因突变体 来帮助研究伯氏疏螺旋体 等病原体8。使用人工喂养系统有助于研究人员通过允许感染或传播只发生在蜱虫一侧来隔离基因的作用，从而隔离可能混淆此类研究的任何宿主反应。
同样，与疾病和动物传播有关的蜱虫的某些生命阶段可能不会被诱导以常见的实验室模式物种为食。例如， 肩突硬蜱 雌性必须以较大的动物为食，通常是兔子9。虽然通常可用于实验室实验，但使用兔子的行政和管理要求超过了小型啮齿动物的要求，并且对于某些实验室来说可能令人望而却步。其他蜱虫种类，特别是那些兽医关注的蜱虫物种，必须以牛或其他大型动物为食，这些动物在大多数实验室中都不实用。 体外 喂养和感染方法，如人工膜喂养，为使用大型或外来宿主动物提供了替代方案。
此外，使用人工饲喂系统可以进行某些传统动物饲喂方法无法实现的分析。一个这样的例子是，通过将血源与喂养机制分离，可以检查不同宿主的血液在伯氏疏螺旋体传播中的作用10。这种对宿主血液的检查以及血液本身在没有宿主免疫反应的情况下所起的作用是能够理解病原体传播周期的重要因素，也是人工喂养系统能够帮助回答的问题11。还可以在饲料期间量化病原体的确切传播次数，而不仅仅是检查宿主中的传播成功和建立8，12。
在 1950 年代和 1960 年代，一些用于硬蜱的第一批人工摄食膜是由动物皮或动物源性膜制成的13，14。由于这些膜的生物学性质，新膜的生产和保质期都存在问题。在 1990 年代，开发了全人造膜，它利用网、纸或织物的背衬和硅胶浸渍15，16。有机硅是理想的选择，因为它的物理特性模仿了皮肤的弹性和轻微的粘性，以及它的生物固有特性。在此基础上，Krober 和 Guerin（该技术所基于的工作）描述了一种用于人工喂养 I. ricinus17 的有机硅浸渍人造丝膜喂养技术。
肩胛骨弧菌（一种密切相关的物种）方法的改进导致膜浸渍中使用的有机硅硬度、膜生产配方、腔室尺寸和附着兴奋剂存在显着差异。虽然本研究中报告的改进导致了与Andrade等人报告的膜特性相似的膜特性，他还开发了基于Krober和Guerin的有机硅膜用于肩胛骨I.，有机硅浸渍步骤存在差异，这使得可以灵活地将该方案用于肩胛骨I.的未成熟生命阶段15，18.本研究还描述了基于重复使用此方法的添加和技术更改、导致成功源的最佳实践以及可能出现的问题的故障排除。这种方法已被用于喂养所有活跃的生命阶段，用致病菌感染蜱虫，并将蜱虫暴露于多种剂量的抗生素19，20。虽然所示的人工膜喂养方法是针对肩胛蜱的，但这种方法很容易适应其他种类的蜱，膜厚度略有改变。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>00-10 Hardness Silicone</td>
			<td>Smooth-On</td>
			<td>Ecoflex 00-10</td>
			<td>Trial size from Smooth-On Store</td>
		</tr>
		<tr>
			<td>00-50 Hardness Silicone</td>
			<td>Smooth-On</td>
			<td>Ecoflex 00-50</td>
			<td>Trial size from Smooth-On Store</td>
		</tr>
		<tr>
			<td>30 Hardness Silicone</td>
			<td>Smooth-On</td>
			<td>Mold Star 30</td>
			<td>Trial size from Smooth-On Store</td>
		</tr>
		<tr>
			<td>6-well cell culture plates</td>
			<td>Corning Incorporated</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenosine triphosphate (ATP)</td>
			<td>Millipore Sigma</td>
			<td>A1852-1VL</td>
			<td>Used to make an aqueous solution of 3 mM ATP that has been filter sterlized via 0.2 micometer filter</td>
		</tr>
		<tr>
			<td>Bovine blood</td>
			<td>HemoStat</td>
			<td>DBB500</td>
			<td>Mechanically defibrinated; 500 mL is usually sufficient for one experiment</td>
		</tr>
		<tr>
			<td>Clingwrap</td>
			<td>Fisherbrand</td>
			<td>22-305654&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter Paper</td>
			<td>Fisherbrand</td>
			<td>09-790-2C</td>
			<td>Autoclave and let cool before using. Can use Fine quality instead of medium too</td>
		</tr>
		<tr>
			<td>Fluon (aqueous polytetrafluoroethylene)</td>
			<td>Bioquip</td>
			<td>2871</td>
			<td>Available from other sources such as https://canada-ant-colony.com/products/fluon-ptfe-10ml</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Millipore Sigma</td>
			<td>G8270-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Hexane</td>
			<td>Millipore Sigma</td>
			<td>139386-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Lens paper</td>
			<td>Fisherbrand</td>
			<td>11-995</td>
			<td>100% rayon</td>
		</tr>
		<tr>
			<td>Nystatin &#160;</td>
			<td>Gold Biotechnology</td>
			<td>N-750-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Fisherbrand</td>
			<td>S37440&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin/fungizone</td>
			<td>Gibco</td>
			<td>15240-096</td>
			<td>Or equivalent generic with concentration as follows (10,000 units/mL of penicillin, 10,000 &#181;g/mL of streptomycin, and 25 &#181;g/mL of Amphotericin B)</td>
		</tr>
		<tr>
			<td>Phagostimulant</td>
			<td>Made in House</td>
			<td></td>
			<td>Collected from prior tick feeds</td>
		</tr>
		<tr>
			<td>Polycarbonate Pipe</td>
			<td>McMaster-Carr</td>
			<td>8585K204&#160;</td>
			<td>Cut to 45 mm length, 1.25 inch outer diameter, 1 inch inner diameter. Cutting requires a chop saw grinding wheel.</td>
		</tr>
		<tr>
			<td>Rubber O-rings</td>
			<td>McMaster-Carr</td>
			<td>9452K38&#160;</td>
			<td>5 mm thick, 1.25 inch inner diameter</td>
		</tr>
		<tr>
			<td>Soft touch forceps</td>
			<td>VWR</td>
			<td>470315-238&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Super glue</td>
			<td></td>
			<td></td>
			<td>cyanoacrylate glue</td>
		</tr>
		<tr>
			<td>Unryu paper&#160;</td>
			<td>Art supply stores</td>
			<td></td>
			<td>mulberry fiber 10 g/m2. Purchased at Wet Paint art supply store, St. Paul, MN, USA</td>
		</tr>
	</tbody>
</table>

tick artificial membrane feeding for ixodes scapularis

蜱虫及其相关疾病由于其公共卫生和兽医负担而是一个重要的研究课题。然而，在研究和饲养过程中对蜱虫的喂养要求可能会限制实验问题或实验室研究蜱虫及其相关病原体的能力。人工膜饲喂系统可以减少这些问题，并开辟传统动物饲喂系统可能无法实现的新研究途径。本研究描述了一种人工膜喂养系统，该系统已经过改进，可在所有 肩突硬蜱 生命阶段成功进食和肿胀。此外，本研究中描述的人工膜喂养系统可以通过简单细化所需的膜厚度来修改以用于其他蜱虫物种。人工膜饲喂系统的好处被系统的劳动密集型、可能影响饲喂成功的其他环境因素以及针对蜱虫的每个新物种和生命阶段改进技术的需求所抵消。

成功的喂养取决于是否需要部分或完全充血。成功喂养的 肩胛骨I. 在成虫中变成青铜灰色，并自行从膜上分离。但是，如果它们至少是豌豆大小的，则在完成喂食时，它们可能会从膜上分离。对于 肩蜛不成熟 阶段，完全充血的蜱的大小各不相同，并且由于与成虫不同，它们不会表现出颜色变化，因此脱离是确定蜱是否完全充血的最佳方法。参见 图3 ，了解若?...

Watch this Scientific Journal Video about Tick Artificial Membrane Feeding for Ixodes scapularis at JoVE.com

Tick Artificial Membrane Feeding for Ixodes scapularis

这里介绍的是一种通过人工膜系统 在体外对 蜱虫进行血液喂养的方法，以允许各种蜱生命阶段的部分或完全充血。

肩突硬蜱人工膜喂养

Ticks and their associated diseases are an important topic of study due to their public health and veterinary burden. However, the feeding requirements of ...

虽然以前已经进行过人工膜喂养，但我们的方案更容易设置，并且通过一些修改即可适应其他蜱虫物种。人工膜喂养的主要优点是它允许暴露于病原体和通过动物喂养可能无法实现的治疗方法。这种人工膜进料系统可以应用于其他节肢动物物种，对理想的吞噬刺激剂进行一些测试，并对理想的膜厚度进行一些修改。生产正确的膜厚度需要一些练习。确保使用千分尺测量膜上多个位置的厚度。我们实验室的博士后Benjamin Cull将帮助我演示该程序。首先，用70%乙醇擦拭平坦的无孔表面，例如臂架的玻璃板或陶瓷涂层金属底座，然后用单层保鲜膜覆盖。确保保鲜膜平整，没有气泡或皱纹。将100%人造丝镜头清洁纸胶带到准备好的表面上。确保它平坦并用胶带在纸张的所有四个面上略微拉紧。通过将硅胶套件的每个部分的五毫升量出五毫升到一次性容器中来制备 0010 硬度硅胶混合物。将两种液体轻轻混合，加入1.5毫升己烷。继续混合，直到混合物均匀并充分混合。使用小刮刀将硅胶混合物分布在镜头纸上，静置一分钟，以确保硅胶已浸入镜头纸中。稳定地，使用少量向下压力用刮刀将多余的硅胶刮到一边。用刮刀进行第二次通过，不要施加向下的压力以去除任何硅胶线并产生光滑的层。仅对于幼虫，将饲喂室的顶部浸入Fluon含氟聚合物树脂中数次，使其在两次施用之间干燥以产生一致的层。让膜在无尘环境中固化至少 24 小时，例如封闭的化学品或生物安全柜。接下来，混合等量的有机硅混合物A和B，以制备30硬度的硅胶混合物，以将腔室连接到膜上。将聚碳酸酯腔室浸入硅胶混合物中约四分之一英寸，然后将它们放在膜片上，注意不要与任何胶带重叠。让附着硅胶固化至少 24 小时。使用手术刀，小心地切割每个连接的腔室周围的膜，修剪边缘，使其可以顺利地装入六孔板的孔中，而不会在侧面刮擦太多。在腔室内留出足够的材料，以最大限度地添加膜。从膜片的每个角和中心切下一小块剩余的膜。从每块上取下保鲜膜，用千分尺测量塑料片以确定平均膜厚度。将喂料室与每个室的一个 O 形圈一起放入玻璃烧杯中，在 121 摄氏度下高压灭菌至少 20 分钟以消毒。使用前让腔室冷却。取出预先准备好的高压灭菌膜室并将O形圈放在它们周围后，在每个腔室中填充足够的70%乙醇以覆盖膜。让它在六孔板中静置五分钟，然后寻找腔室和膜之间以及膜本身中的任何泄漏。从腔室中清空乙醇，然后在生物安全柜或层流罩内风干。要加热和激活血液中的补体，请在 56 摄氏度下加热 40 分钟，并用每升葡萄糖 2 克补充机械除颤牛血。膜干燥后，将约20微升吞噬兴奋剂涂抹在腔室内部，并倾斜腔室以将其散布在表面周围。当吞噬兴奋剂干燥时，用刷子或镊子快速将蜱虫添加到腔室中。用封口膜密封顶部，因为水浴的热量会导致它撕裂。然后将每个孔或腔室中加入五毫升的热量和活化血液到锥形管中，并将其放入设置在36摄氏度的水浴中。每5毫升血液中加入5微升3毫摩尔ATP和50微升100X青霉素链霉素真菌区库存，并将4.5毫升血液转移到六孔板的孔中。轻轻地将腔室放入孔中，调整腔室上的O形圈高度，使膜位于血液中，但侧面周围的血液水平不会超过孔。然后将孔以一定角度放入血液中，以避免血液和膜之间形成气泡。将六个孔板的盖子放在喂养室的顶部。将带有腔室的六孔板放入准备好的34摄氏度水浴和盖子中。每孔加入5微升3毫摩尔ATP和50微升100X青霉素链霉素真菌区库存到血管中，并将4.5毫升血液转移到新鲜六孔板的孔中，如前所述。从水浴中取出带有滴答室的六孔板，然后将室从孔中取出。用10毫升灭菌的1X PBS冲洗腔室和膜的外部以去除血液。使用高压灭菌的滤纸，轻轻擦干膜和腔室以去除多余的PBS。如果腔室内有大量冷凝水，请先用高压灭菌的滤纸轻拍干燥湿点，然后再用新鲜的封口膜密封。然后更换腔室顶部的封口膜。将蜱虫室放入新的六孔板中，并根据需要调整O形圈高度。对板上的每个腔室重复这些步骤。最后，将六孔板的盖子放在腔室顶部，然后将新的六孔板移回34摄氏度的水浴中。此处显示了肩胛骨若虫部分充血的持续饲料。附着部位周围的黑色或棕色点是在饲料期间和之后可以收集的霜冻，以制造吞噬兴奋剂。部分充血的肩胛骨若虫附着在膜上。在这里也可以看到充血的幼虫霉菌的外壳。蜱虫充血数和种蛋质量重量在此表中列出。幼虫和若虫充血实验条件除了该技术中使用的补充剂外，还向血液中添加了鹿器官匀浆。在这里，成功的充血被定义为成功蜕皮到下一个活阶段的充血蜱虫或成功产卵的充血雌性。将硅胶涂在镜片纸上时，需要练习才能获得可接受的厚度，因此在使用前测量膜的厚度始终很重要。膜喂养会使蜱暴露给病原体或抗生素。这些对蜱生物学方面的影响，如蜕皮成功、产卵和存活率，可以在饲料后进行评估。

Research

JoVE Journal

Biology

Tick Artificial Membrane Feeding for Ixodes scapularis

Testing Nicotine Tolerance in Aphids Using an Artificial Diet Experiment

使用人工饲料实验测试蚜虫的尼古丁公差

Plants may upregulate the production of many different seconday metabolites in response to insect feeding. One of these metabolites, nicotine, is well ...

科研

生物学

Preparation of Artificial Bilayers for Electrophysiology Experiments

电生理实验人工双层膜的制备

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. These bilayers can ...

Crystallizing Membrane Proteins for Structure Determination using 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

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

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 ...

Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery

微灌技术在调查微血管渗透性的大鼠肠系膜条例

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular ...

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

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

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 ...