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

Yazarlar, tavsiye ve reaktifler için Anna Walduck Christina Cheers, Stuart Berzins, Dale Godfrey, Yifan Zhan ve Jonathan Wilksch teşekkür etmek istiyorum. Bu çalışma Juvenil Diyabet Araştırma Vakfı (1-2008-602) ve Avustralya Ulusal Sağlık ve Tıbbi Araştırma Konseyi (575.552) tarafından finanse edildi. KB, bir Avustralyalı Lisansüstü Ödülü tarafından desteklenmektedir. OW Avustralya Ulusal Sağlık Medikal Araştırma Konseyi, bir RD Wright Kardeşliği tarafından desteklenmektedir.

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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>

Çıkar çatışması ilan etti.

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> Reaktifin Adı </th><th> Şirket </th><th> Katalog numarası </th><th> Yorumlar </th></tr><tr><td> At kanlı agar baz No.2 </td><td> Oxoid </td><td> CM0271 </td><td> HBA plakalar için agar tabanının hazırlanması </td></tr><tr><td> At kan (defibrine) </td><td> Oxoid </td><td> HB1000 </td><td> Agar baz% 5-10 </td></tr><tr><td> Beyin kalp infüzyonu (BHI) suyu (10 ml) </td><td> Oxoid </td><td> TM456 </td><td></td></tr><tr><td> Beyin kalp infüzyon susuz medya </td><td> Oxoid </td><td> CM1135 </td><td></td></tr><tr><td> 96-düz alt plakası </td><td> BD Biosciences </td><td> 353072 </td><td></td></tr><tr><td> 70 mikron hücre süzgecinden </td><td> BD Biosciences </td><td> 352350 </td><td></td></tr><tr><td> Küçük petri </td><td> BD Biosciences </td><td> 351007 </td><td></td></tr><tr><td> Stomacher 80 Biomaster laboratuarı sistemi </td><td> Seward </td><td></td><td></td></tr><tr><td> Plastik Stomacher çanta </td><td> Sarstedt </td><td> 86 9924 530 </td><td></td></tr><tr><td> Sığır serum albumini </td><td> Sigma </td><td> A3912 </td><td></td></tr><tr><td> FACS tampon </td><td></td><td></td><td> % 0,1 (w / v) PBS içinde sığır serum albumin </td></tr><tr><td> İzotonik Percoll (33.75% Percoll PBS içinde) </td><td> GE Healthcare </td><td> 17-0891-01 </td><td> 33.75mL Percoll 3.75mL 10x PBS, 62.5mL 1x PBS 100 ml izotonik Percoll yapar </td></tr><tr><td> TAC Tampon </td><td></td><td></td><td> 17mm Tris, distile su ile 140 amonyum klorür </td></tr><tr><td> FCS / EDTA tampon </td><td></td><td></td><td> 10mM EDTA ile fetal buzağı serumu </td></tr><tr><td> FACS / EDTA tampon </td><td></td><td></td><td> FACS tampon + 5mM EDTA </td></tr><tr><td> Tripan mavi </td><td> Sigma </td><td> T6146 </td><td> PBS içinde% 0.4 (w / v), filtre steril </td></tr><tr><td> 2mL Cryovial </td><td> Greiner Bio One </td><td> 121263 </td><td></td></tr><tr><td> 27 gauge/1mL insülin enjektörü </td><td> Terumo Tıbbi Ürünler </td><td> SS10M2713 </td><td></td></tr><tr><td> Iğne </td><td> Terumo Tıbbi Ürünler </td><td> NN-2516R (25G 5/8in) NN-2613R (26g 1/2in) </td><td></td></tr><tr><td> Şırınga </td><td> Terumo Tıbbi Ürünler </td><td> SS-01T (1 ml), SS-053 (5ml), SS-10S (10 ml) </td><td></td></tr></tbody></table>

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

Listeria monocytogenes (Listeria) Gram-pozitif fakültatif hücre içi bir patojen 1. Fareler üzerinde yapılan çalışmalarda genellikle intravenöz enjeksiyon Listeria istihdam, sistemik enfeksiyon 2 sonuçlanır. Enjeksiyondan sonra, Listeria CD8α + dendritik hücreleri ve Kupffer hücreleri 3,4 alımı nedeniyle dalak ve karaciğer hızla yaymaktadır. Bir kez fagosite, çeşitli bakteriyel proteinler, phagosome kaçmak, sitoplazmada içinde hayatta ve komşu hücreleri enfekte 5 Listeria sağlar . Enfeksiyon ilk üç gün boyunca, farklı doğuştan gelen bağışıklık hücreleri (örneğin monositler, nötrofil, NK hücreleri, dendritik hücreler) Listeria çoğalması en aza indirmek bakterisidal mekanizmalar aracılık. CD8 + T hücreleri, daha sonra işe ve nihai temizlenmesi için genellikle 10 gün enfeksiyonu 6 içinde ev sahibi, Listeria sorumludur . Başarılı klerensi enfekte olmuş fareler Listeria konağın immün yanıtları 6 uygun başlangıçlı bağlıdır. Kendilenmiş fare suşlarının 7,8 arasından geniş bir hassasiyetleri vardır. Genellikle, Listeria enfeksiyonuna yatkınlığı olan farelerin daha az dayanıklı farelere kıyasla artmış bakteri yükü ve / veya gecikmiş boşluk gösteren bakteri çoğalması kontrol edebiliyoruz . Bağlantı analizleri ve nakavt fare suşları da dahil olmak üzere genetik çalışmalar, çeşitli gen dizisi varyasyon Listeria enfeksiyonu 6,8-14 ev sahipliği yanıtları etkiler var. Bu nedenle, farklı fare suşları arasında enfeksiyon kinetiği belirlenmesi ve karşılaştırılması Listeria karşı bağışıklık yanıtlarını katkıda konak genetik faktörlerin belirlenmesi için önemli bir yöntemdir. Konakçı tepkileri farklı Listeria suşlarının karşılaştırılması da antibiyotik tedavisi ya da aşısı tasarımı için potansiyel hedefler olarak hizmet verebilir bakteriyel virülans faktörleri tanımlamak için etkili bir yoldur . Biz burada bakteri yükü (koloni oluşturan birim [CFU] başına doku) ölçme ve Listeria ile enfekte farelerde bağışıklık yanıtları FACS analizi için karaciğer ve dalak tek hücre süspansiyonlarının hazırlanması için basit bir yöntem açıklanmaktadır. Bu yöntem ilk romanı fare suşlar Listeria enfeksiyonu karakterizasyonu, yanı sıra bağışıklık yanıtlarını, Listeria ile enfekte olan farklı fare suşları arasında karşılaştırma için özellikle yararlıdır . Biz, kan agar kültür, β-hemoliz 1 (Şekil 1) nedeniyle her koloninin çevresinde karakteristik bir hale bölge sergiler suşu 15 EGD Listeria monocytogenes kullanın. Bakteriyel yük ve bağışıklık yanıtlarını aşağıda açıklanan protokolleri kullanarak FACS analizi için kan agar plakalarını ve doku hücre süspansiyonlarının hazırlanması kültür dokusu Homojenat enfeksiyonu sonra herhangi bir zaman noktasında tespit edilebilir. Biz, immün sistemi baskılanmış veya hamile bireyler Listeria işlemez gerektiğine dikkat cekti ve ilgili kurumsal biyogüvenlik komitesi ve hayvan tesis yönetimi, işe başlanmadan önce danışılmalıdır .

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

Listeria monocytogenes farelerde bağışıklık yanıtlarını ve genetik yatkınlık bakterilerin hücre içi eğitim için bir model organizmadır. Bu yöntem, bakteri yükü ölçmek ve Listeria enfeksiyonu nedeniyle bağışıklık hücreleri değişiklikleri belirlemek için FACS analiz için farelerin karaciğer ve dalak tek hücre süspansiyonları oluşturmak için bir olanak sağlar.

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