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

저자는 조언과 시약에 대한 안나 Walduck, 크리스티나 건배, 스튜어트 Berzins, 데일 고드프리, Yifan Zhan와 조나단 윌크쉬 감사하고 싶습니다. 이 작품은 소아 당뇨병 연구 재단 (1-2008-602)과 호주 국립 보건 의학 연구위원회 (575552)에 의해 재정 지원되었다. NW는 호주 대학원 수상에 의해 지원됩니다. 아우는 호주 국민 건강 의학 연구위원회에서 RD 라이트 원정대에 의해 지원됩니다.

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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>

관심 없음 충돌 선언하지 않습니다.

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> 시약의 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th><th> 댓글 </th></tr><tr><td> 호스 피 한천 기지 제 2 </td><td> Oxoid </td><td> CM0271 </td><td> HBA 플레이트에 대한 한천 기지의 준비 </td></tr><tr><td> 호스 피 (defibrinated) </td><td> Oxoid </td><td> HB1000 </td><td> 한천베이스 50~10% 추가 </td></tr><tr><td> 뇌 심장 주입 (BHI) 국물 (10 ML) </td><td> Oxoid </td><td> TM456 </td><td></td></tr><tr><td> 뇌 심장 주입 탈수 미디어 </td><td> Oxoid </td><td> CM1135 </td><td></td></tr><tr><td> 96 - 웰 플랫 바텀 플레이트 </td><td> BD Biosciences </td><td> 353072 </td><td></td></tr><tr><td> 70 μm의 셀 스트레이너 </td><td> BD Biosciences </td><td> 352350 </td><td></td></tr><tr><td> 소형 페트리 접시 </td><td> BD Biosciences </td><td> 351007 </td><td></td></tr><tr><td> Stomacher 80 biomaster 실험실 시스템 </td><td> 슈어드 </td><td></td><td></td></tr><tr><td> 플라스틱 Stomacher 가방 </td><td> Sarstedt </td><td> 86 9,924 530 </td><td></td></tr><tr><td> 소 혈청 알부민 </td><td> 시그마 </td><td> A3912 </td><td></td></tr><tr><td> 외과 버퍼 </td><td></td><td></td><td> 0.1 % (W / V) PBS의 소 혈청 알부민 </td></tr><tr><td> Isotonic Percoll (PBS에서 33.75 %의 Percoll) </td><td> GE 헬스케어 </td><td> 17-0891-01 </td><td> 33.75mL Percoll, 3.75mL 10X PBS는 62.5mL 1X PBS는 100mL isotonic Percoll하게 </td></tr><tr><td> TAC 버퍼 </td><td></td><td></td><td> 17mM 트리스, 증류수에 140mM 암모늄 클로라이드 </td></tr><tr><td> FCS / EDTA (에틸렌 다이아 민 테트라 초산) 버퍼 </td><td></td><td></td><td> 10mM EDTA (에틸렌 다이아 민 테트라 초산)과 태아 송아지 혈청 </td></tr><tr><td> 외과 / EDTA (에틸렌 다이아 민 테트라 초산) 버퍼 </td><td></td><td></td><td> 외과 버퍼 + 5mM EDTA (에틸렌 다이아 민 테트라 초산) </td></tr><tr><td> Trypan 파랑 </td><td> 시그마 </td><td> T6146 </td><td> PBS에 0.​​4 % (W / V), 필터는 살균 </td></tr><tr><td> 2mL Cryovial </td><td> 그라 이너 바이오 원 </td><td> 121263 </td><td></td></tr><tr><td> 27 gauge/1mL 인슐린 주사기 </td><td> Terumo 의약품 </td><td> SS10M2713 </td><td></td></tr><tr><td> 바늘 </td><td> Terumo 의약품 </td><td> NN - 2516R (25G 5/8in) NN - 2613R (26G 1/2in) </td><td></td></tr><tr><td> 주사기 </td><td> Terumo 의약품 </td><td> SS - 01T (1mL), SS - 053 (5mL), SS - 10 초 (10mL) </td><td></td></tr></tbody></table>

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

리스테리아 monocytogenes는 (리스테리아) 그람 양성 통성 세포내 병원체 1입니다. 마우스 연구는 일반적으로 리스테리아의 정맥 주사를 채용, 어떤은 조직의 감염이 초래합니다. 주입 후, 리스테리아 신속하게 CD8α + 돌기 세포와 Kupffer 세포 3,4에 의해 이해로 인해 비장과 간장에 disseminates. 일단 phagocytosed, 각종 세균의 단백질은 phagosome 탈출 cytosol 내에 생존하고, 인접 세포에게 5 리스테리아 감염 수 있습니다. 감염의 처음 세 일 동안, 다른 타고난 면역 세포 (예 : monocytes, neutrophils, NK 세포, 세포 돌기) 리스테리아 확산을 최소화 bactericidal 메커니즘을 중재. CD8 + T 세포는 이후 모집 일반적으로 감염 6 10 일 이내에 호스트에서 리스테리아의 최종 통관에 대한 책임이 있습니다. 감염된 생쥐에서 리스테리아의 성공적인 허가는 호스트 면역 반응 6 해당 증상에 따라 달라집니다. 근친 마우스 종자 7,8 사이에 민감한 다양한있다. 일반적으로, 리스테리아 감염 증가 느끼기 쉬운 상태로 마우스는 마우스에 비해 저항력이 증가 세균 하중 및 / 또는 지연 허가를 보여주는, 박테리아 증식을 제어하는 덜 수 있습니다. 결합 분석 및 녹아웃 마우스 종자를 포함한 유전자 연구, 시퀀스 유사 리스테리아 감염 6,8-14에 호스트 응답에 영향을 미치는있는 다양한 유전자를 발견했습니다. 결단력과 다른 마우스 변종 사이의 감염 속도론의 비교 그러므로 리스테리아에 대한 면역 반응에 기여하는 호스트 유전 요소를 식별하는 중요한 방법입니다. 다른 리스테리아 변종에 호스트 응답의 비교도 항생제 치료 또는 예방 접종 디자인에 대한 잠재적인 대상으로 될 수있다 세균성 독성 요소를 식별하는 효과적인 방법입니다. 우리는 여기에 세균 하중 (콜로니 형성 단위 [CFU] 당 조직)을 측정하고 리스테리아에 감염된 생쥐에서 면역 반응의 외과의 분석을위한 간 및 비장의 단일 셀 suspensions을 준비를위한 간단한 방법을 설명합니다. 이 방법은 초기 소설 마우스 종자에 리스테리아 감염의 특성뿐만 아니라, 리스테리아에 감염된 다른 마우스 종자 간의 면역 반응 비교에 특히 유용합니다. 우리는 혈액 한천에 교양하면 β - 용혈 1 (그림 1)로 인해 각각의 콜로니 주위 특성 헤일로 영역을 전시되는 변형율 15 EGD 리스테리아 monocytogenes를 사용합니다. 박테리아로드하고 면역 반응은 아래에 설명된 프로토콜을 사용하여 외과 분석을위한 혈액 한천 플레이트 및 준비 조직 세포 suspensions에 culturing 조직 homogenate에 의해 감염 후 언제든지 포인트에서 확인할 수 있습니다. 우리는 immunocompromised 또는 임신 개인 리스테리아 처리하지 말아야합니다 것이며, 작업 개시 전에 관련 기관 biosafety위원회 동물 시설 관리 자문해야합니다.

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

리스테리아 monocytogenes는 생쥐에서 세균을 세포에 면역 반응과 유전 자화율을 연구를위한 모델 생물이다. 이 방법은 박테리아 부하를 측정하고 리스테리아 감염으로 인한 면역 세포의 변화를 결정하는 외과 분석을위한 생쥐의 간 및 비장의 단일 셀 suspensions를 생성하는 하나 있습니다.

감염된 마우스에 세균 하중과 면역 응답 측정 리스테리아 monocytogenes</em

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

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

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유동세포계측법로 고정하고 살아있는 세포에서 Calpain 활동을 측정

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

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Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

경구 전송<em&gt; 리스테리아</em&gt; 오염 된 음식의 섭취를 통해 마우스의

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the ...

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

세균성 병원체에 의해 세포 감염을 조사하기 위해 InlC 분비 이미징<em&gt; 리스테리아</em

Bacterial intracellular  pathogens can be conceived as molecular tools to dissect cellular signaling  cascades due to their capacity to exquisitely ...

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

리스테리아 단세포 유전자를 사용하여 감염된 마우스에 있는 문화와 심혼 식민지에 있는 심장 세포의 세균성 침략 평가

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised ...

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

<em&gt; 체외</em&gt; HIV 공동 감염의 맥락에서 말라리아에 면역 반응을 측정하기위한 모델

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

RNA 정제는 세포 내에서 재배<em&gt; 리스테리아</em&gt; 식세포 세포에

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. ...

Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-&#947; Responses

Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â³ Responses

와 실험 감염<em&gt; 리스테리아</em&gt; 호스트 인터페론-γ 응답 유학을위한 모델로

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

살 모 넬 라 typhimurium의 절연-대 식 세포에서 Phagosomes를 포함 하

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

감염 된 Ifnar1/- 생쥐의 Immunoprivileged 장기에서 Zika 바이러스 관련 T 세포 응답의 평가

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...

Listeria monocytogenes Infection of the Brain

Listeria monocytogenes Infection of the Brain

Listeria monocytogenes 뇌의 감염

Listeria monocytogenes is an intracellular bacterial pathogen that is frequently associated with food-borne infection. The ability of&#160;L. ...