Name: measuring bacterial load and immune responses in mice infected with listeria monocytogenes
Uploaded: 2011-08-09T13:00:00
Duration: 12 min 50 s
Description: Watch this Scientific Journal Video about Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes at JoVE.com

作者想感谢安娜Walduck，克里斯蒂娜欢呼声，司徒贝尔津什，戴尔戈弗雷，一帆詹和乔纳森Wilksch意见和试剂。这项工作是由青少年糖尿病研究基金会（1-2008-602）和澳大利亚国家卫生和医学研究理事会（575552）的资助。净重是支持澳大利亚研究生奖。单程是由RD莱特奖学金由澳大利亚国家卫生医学研究理事会的支持。

<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=Multigenic+control+of+Listeria+monocytogenes+susceptibility+in+mice.">Multigenic control of Listeria monocytogenes susceptibility in mice.</a> Nat Genet. 27, 259-260 (2001).</li><li>Boyartchuk, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+host+resistance+locus+sst1+controls+innate+immunity+to+Listeria+monocytogenes+infection+in+immunodeficient+mice.">The host resistance locus sst1 controls innate immunity to Listeria monocytogenes infection in immunodeficient mice.</a> J Immunol. 173, 5112-5120 (2004).</li><li>Pan, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ipr1+gene+mediates+innate+immunity+to+tuberculosis.">Ipr1 gene mediates innate immunity to tuberculosis.</a> Nature. 434, 767-772 (2005).</li><li>Garifulin, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Irf3+polymorphism+alters+induction+of+interferon+beta+in+response+to+Listeria+monocytogenes+infection.">Irf3 polymorphism alters induction of interferon beta in response to Listeria monocytogenes infection.</a> PLoS Genet. 3, 1587-1597 (2007).</li><li>Hof, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virulence+of+different+strains+of+Listeria+monocytogenes+serovar+1/2a.">Virulence of different strains of Listeria monocytogenes serovar 1/2a.</a> Med Microbiol Immunol (Berl. 173, 207-218 (1984).</li><li>Wollert, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extending+the+host+range+of+Listeria+monocytogenes+by+rational+protein+design.">Extending the host range of Listeria monocytogenes by rational protein design.</a> Cell. 129, 891-902 (2007).</li><li>Lecuit, 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+transgenic+model+for+listeriosis:+role+of+internalin+in+crossing+the+intestinal+barrier.">A transgenic model for listeriosis: role of internalin in crossing the intestinal barrier.</a> Science. 292, 1722-1725 (2001).</li><li>Marco, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Penetration+of+Listeria+monocytogenes+in+mice+infected+by+the+oral+route.">Penetration of Listeria monocytogenes in mice infected by the oral route.</a> Microb Pathog. 23, 255-263 (1997).</li><li>Busch, D. H., Vijh, S., Pamer, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+model+for+infection+with+Listeria+monocytogenes.">Animal model for infection with Listeria monocytogenes.</a> Curr Protoc Immunol. Chapter. 19, 19-19 (2001).</li><li>Neuenhahn, M., Schiemann, M., Busch, 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=DCs+in+mouse+models+of+intracellular+bacterial+infection.">DCs in mouse models of intracellular bacterial infection.</a> Methods Mol Biol. 595, 319-329 (2010).</li><li>Conlan, J. W., North, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophils+are+essential+for+early+anti-Listeria+defense+in+the+liver,+but+not+in+the+spleen+or+peritoneal+cavity,+as+revealed+by+a+granulocyte-depleting+monoclonal+antibody.">Neutrophils are essential for early anti-Listeria defense in the liver, but not in the spleen or peritoneal cavity, as revealed by a granulocyte-depleting monoclonal antibody.</a> J Exp Med. 179, 259-268 (1994).</li><li>Czuprynski, C. J., Brown, J. F., Wagner, R. D., Steinberg, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Administration+of+antigranulocyte+monoclonal+antibody+RB6-8C5+prevents+expression+of+acquired+resistance+to+Listeria+monocytogenes+infection+in+previously+immunized+mice.">Administration of antigranulocyte monoclonal antibody RB6-8C5 prevents expression of acquired resistance to Listeria monocytogenes infection in previously immunized mice.</a> Infect Immun. 62, 5161-5163 (1994).</li></ol>

没有利益冲突的声明。

Listeria is one of the most widely used organisms to characterize host immune responses to intracellular bacteria6. The protocol presented here enables one to measure bacterial load and immune cell responses within the same tissue of a given mouse. This dual measurement of a particular tissue for each infected mouse provides for more robust comparisons within and between mouse cohorts (either representing different mouse strains or time points post-infection). While Listeria infection by oral administration could also be used to study immune responses in mice, infection by intravenous injection is often used because: 1) it ensures rapid and effective delivery to the bloodstream; 2) it results in a synchronized and consistent systemic infection; and 3) the Mus species harbors a mutation in the gene encoding the E-cadherin receptor, which limits Listeria infection by oral administration (this mutation affects Listeria's ability to bind the mouse E-cadherin receptor and to efficiently cross the epithelial lining of the gastrointestinal tract)16-18.
There are a few critical steps in this protocol. First, the Listeria inoculum stock should be generated from a fresh overnight HBA culture to ensure viability and virulence. Second, it is important to accurately determine the CFU concentration of the inoculum before and after injecting all mice to ensure that the CFU concentration does not differ greatly between the first and last mouse injected. Third, injection of Listeria into the tail vein must be consistent for all mice. Lastly, it is necessary to perfuse the liver to deplete non-resident leukocytes to ensure accurate measurement of immune cells within the liver and not leukocytes passing through in the peripheral blood. All of these steps, if not performed successfully, can result in unwanted variability for bacterial load and/or immune responses between individual mice infected with Listeria. 
Two limitations of this protocol are the investigator's skill for infecting mice by intravenous injection and the detection of bacterial load in tissues. If one person is injecting a large number of mice, Listeria viability (i.e. CFU concentration) may reduce over time once the frozen inoculum is thawed (e.g. &gt;2 hours between injecting first and last mouse). It is up to the investigator to determine how many mice she/he can inject before the inoculum is compromised. Another limitation of this method is that the Listeria load cannot be accurately measured below 100CFU/organ due to the relatively small amounts of cultured tissue homogenate (e.g. Figure 3 indicates that 100CFU/organ is the detection limit). To more accurately measure lower values for CFU/organ, a larger amount of the tissue homogenate can be cultured (up to 0.5mL per plate, multiple plates can be used) so a greater proportion of the tissue is sampled for detection of Listeria. If a Stomacher is not available, then alternative methods to homogenate the tissue, such as using a tissue homogenizer, can be used instead for steps 7.1-7.2. On a practical note, if space in the 37&deg;C incubator is limited for culturing tissue samples from infected mice, then HBA culture plates can be incubated at room temperature for 2-3 days (the colonies will grow slower at room temperature). However, room temperature should not be used when growing cultures for preparation of frozen Listeria stocks.
This protocol provides a basic approach for characterizing Listeria infection in mice and can be used with other Listeria strains besides EGD. In addition to the liver and spleen, single-cell suspensions from lymph nodes can also be generated. In either instance, these single-cell suspensions can be used for a variety of analyses, including measuring immune cell subsets, and in vitro stimulation of sorted immune cells. Once the basic techniques are mastered this protocol can also be modified to isolate Listeria-specific T cells19, characterize dendritic cells20, or perform immune cell depletion at certain time-points after infection21,22 to more thoroughly characterize the immune response in mice infected with Listeria.

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论</th></tr><tr><td>马血琼脂基础第二</td><td> Oxoid公司</td><td> CM0271 </td><td>制备琼脂为HBA板基地</td></tr><tr><td>马血（defibrinated） </td><td> Oxoid公司</td><td> HB1000 </td><td>加入5-10％的琼脂基础。 </td></tr><tr><td> “脑心浸液（BHI）肉汤（10毫升） </td><td> Oxoid公司</td><td> TM456 </td><td></td></tr><tr><td>脑心浸液脱水媒体</td><td> Oxoid公司</td><td> CM1135 </td><td></td></tr><tr><td> 96孔平底板</td><td> BD公司</td><td> 353072 </td><td></td></tr><tr><td> 70微米的细胞过滤器</td><td> BD公司</td><td> 352350 </td><td></td></tr><tr><td>小培养皿</td><td> BD公司</td><td> 351007 </td><td></td></tr><tr><td>抹胸80 biomaster实验室系统</td><td>西沃德</td><td></td><td></td></tr><tr><td>塑料抹胸袋</td><td> Sarstedt </td><td> 86 9924 530 </td><td></td></tr><tr><td>牛血清白蛋白</td><td>西格玛</td><td> A3912 </td><td></td></tr><tr><td>流式细胞仪缓冲区</td><td></td><td></td><td> 0.1％（W / V）牛血清白蛋白的PBS </td></tr><tr><td>等渗Percoll（33.75％Percoll在PBS） </td><td> GE医疗集团</td><td> 17-0891-01 </td><td> Percoll 33.75mL，3.75mL 10X PBS，62.5mL 1X PBS 100毫升等渗Percoll </td></tr><tr><td>交咨会的缓冲区</td><td></td><td></td><td> 17MM三，在蒸馏水中140MM氯化铵</td></tr><tr><td> FCS / EDTA缓冲液</td><td></td><td></td><td>用10mm EDTA小牛血清</td></tr><tr><td>流式细胞仪/ EDTA缓冲液</td><td></td><td></td><td>流式细胞仪缓冲区+ 5MM EDTA </td></tr><tr><td>台盼蓝</td><td>西格玛</td><td> T6146 </td><td> 0.4％，在PBS（W / V），过滤消毒</td></tr><tr><td> 2ML离心管</td><td>格雷纳生物一号</td><td> 121263 </td><td></td></tr><tr><td> 27 gauge/1mL胰岛素注射器</td><td>泰尔茂医疗产品</td><td> SS10M2713 </td><td></td></tr><tr><td>针</td><td>泰尔茂医疗产品</td><td> NN - 2516R（25G 5/8in） NN - 2613R（26G 1/2in） </td><td></td></tr><tr><td>注射器</td><td>泰尔茂医疗产品</td><td> SS - 01T（1ML），SS - 053（5毫升），SS - 10S（10毫升） </td><td></td></tr></tbody></table>

measuring bacterial load and immune responses in mice infected with listeria monocytogenes

李斯特菌（李斯特菌）是革兰氏阳性兼性细胞内病原体1。小鼠研究中，通常采用静脉注射李斯特，这会导致全身性感染 2 。注射后，李斯特的迅速传播到脾脏和肝脏由于摄取CD8α+树突状细胞和Kupffer 细胞 3,4 。一旦吞噬，各种细菌的蛋白质使李斯特逃脱吞噬，在细胞质中求生存，并感染邻近的细胞5。在第3天感染，不同的先天免疫细胞（如单核细胞，中性粒细胞，NK细胞，树突状细胞）介导的杀菌机制，最大限度地减少李斯特菌增殖。 CD8 + T细胞，随后招募和李斯特菌通常在10天感染6的主机，最终清除。 李斯特菌感染小鼠的成功间隙取决于适当的主机 6免疫反应的发病。是一个广泛的敏感性之间自交系小鼠品系7,8。一般来说， 李斯特菌感染的易感性增加的小鼠能够控制细菌繁殖，抗小鼠相比，显示出细菌负荷增加和/或延迟间隙。遗传研究，包括连锁分析和基因敲除小鼠品系，已确定了各种基因序列变异宿主反应，李斯特菌感染 6,8-14的影响。感染不同小鼠品系之间动力学的测定和比较，因此确定主机遗传因素，有助于对李斯特菌的免疫反应的重要方法。宿主反应比较不同的李斯特菌菌株也是一种有效的方法来识别细菌的致病因素，可作为抗生素治疗或疫苗设计的潜在目标。 我们在这里描述一个简单的方法，用于测量细菌（菌落形成单位[CFU]每组织），并准备单细胞悬液，流式细胞仪分析在李斯特菌感染小鼠的免疫反应，对肝，脾。新颖的小鼠品系中的李斯特菌感染的初步鉴定，以及比较不同的小鼠感染李斯特菌株之间的免疫反应，这种方法尤其有用。我们使用的EGD 李斯特菌 15株，血琼脂培养基上培养时，展品周围β-溶血1（图1）由于每个殖民地的特征晕区。可以决定在任何时间点血琼脂平板，并准备组织细胞悬浮液的培养上流式细胞仪分析使用以下协议组​​织匀浆后感染细菌负荷和免疫反应。我们会注意到，那些免疫功能低下或怀孕的人不应该处理李斯特菌，生物安全委员会的有关机构和动物设施管理工作开始前，应征询。

Watch this Scientific Journal Video about Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes at JoVE.com

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

李斯特菌是一种模式生物，为研究免疫反应和遗传易感性细胞在小鼠体内的细菌。这种方法使一个衡量细菌负荷，产生单细胞悬液，流式细胞仪分析小鼠肝和脾，以确定由于李斯特菌感染的免疫细胞的变化。

测量细菌感染小鼠和免疫反应李斯特菌</em

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

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Immunology and Infection

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

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