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

著者らは、アドバイスや試薬のためアンナWalduck、クリスティーナ乾杯、スチュアートBerzins、デイルゴッドフリー、伊帆展とジョナサンWilkschに感謝します。この作品は、少年糖尿病研究財団（1-2008-602）とオーストラリア国立保健医療研究評議会（575552）によって賄われていた。 NWは、オーストラリアの大学院賞でサポートされています。 OWは、オーストラリア国立保健医療研究評議会からRDライトフェローシップでサポートされています。

<ol>
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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>

利害の衝突は宣言されません。

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>試薬の名前</th><th>会社</th><th>カタログ番号</th><th>コメント</th></tr><tr><td>馬血液寒天ベース2号</td><td>オキソイド</td><td> CM0271 </td><td> HBAプレート用寒天ベースの準備</td></tr><tr><td>馬の血（繊維素） </td><td>オキソイド</td><td> HB1000 </td><td>寒天ベースに5〜10％を加算</td></tr><tr><td>ブレインハートインフュージョン（BHI）ブロス（10mL）を</td><td>オキソイド</td><td> TM456 </td><td></td></tr><tr><td>脳心臓浸出液乾燥メディア</td><td>オキソイド</td><td> CM1135 </td><td></td></tr><tr><td> 96ウェル平底プレート</td><td> BDバイオサイエンス</td><td> 353072 </td><td></td></tr><tr><td> 70μmのセルストレーナー</td><td> BDバイオサイエンス</td><td> 352350 </td><td></td></tr><tr><td>小さなペトリ皿</td><td> BDバイオサイエンス</td><td> 351007 </td><td></td></tr><tr><td>ストマッカー80バイオマスターラボシステム</td><td>スワード</td><td></td><td></td></tr><tr><td>プラスチックストマッカー袋</td><td>ザルスタット</td><td> 86 9924 530 </td><td></td></tr><tr><td>ウシ血清アルブミン</td><td>シグマ</td><td> A3912 </td><td></td></tr><tr><td> FACSバッファー</td><td></td><td></td><td> 0.1％（w / v）のPBSに牛血清アルブミン</td></tr><tr><td>等張パーコール（33.75パーセントPBSでパーコール） </td><td> GEヘルスケア</td><td> 17-0891-01 </td><td> 33.75mLパーコール、3.75mL 10倍PBS、62.5mL 1X PBSは、100mLの等張パーコールしています</td></tr><tr><td> TACバッファ</td><td></td><td></td><td> 17mmのトリス、蒸​​留水で140mmの塩化アンモニウム</td></tr><tr><td> FCS / EDTA緩衝液</td><td></td><td></td><td> 10mMのEDTAとウシ胎児血清</td></tr><tr><td> FACS / EDTA緩衝液</td><td></td><td></td><td> FACSバッファ+ 5mMのEDTA </td></tr><tr><td>トリパンブルー</td><td>シグマ</td><td> T6146 </td><td> PBSの0.4％（w / v）の、滅菌フィルタ</td></tr><tr><td> 2mLのクライオバイアル</td><td>グライナーバイオワン</td><td> 121263 </td><td></td></tr><tr><td> 27 gauge/1mLインスリン注射器</td><td>テルモメディカルプロダクツ</td><td> SS10M2713 </td><td></td></tr><tr><td>針</td><td>テルモメディカルプロダクツ</td><td> NN - 2516R（25G 5/8in） NN - 2613R（26G 2インチ） </td><td></td></tr><tr><td>注射器</td><td>テルモメディカルプロダクツ</td><td> SS - 01T（1mLの）、SS - 053（5ml）を、SS - 10S（10ml）を</td><td></td></tr></tbody></table>

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

リステリア菌は、（リステリア）グラム陽性通性細胞内病原体は1です。マウスの研究では、通常、全身感染症2の結果リステリアの静脈内注射を、採用しています。注入後、リステリアはすぐにCD8α+樹状細胞とクッパー細胞3,4による取り込みのために脾臓や肝臓に発信しています。一度貪食、様々な細菌タンパク質は、ファゴソームから逃れるためにリステリアを有効細胞質ゾル内で生き残る、と5隣接する細胞に感染。感染の最初の3日間、別の生来の免疫細胞（例えば単球、好中球、NK細胞、樹状細胞） リステリアの増殖を抑える殺菌のメカニズムを媒介する。 CD8 + T細胞は、その後、通常は感染6の10日間以内に、募集し、ホストからのリステリアの最終的なクリアランスを担当しています。 感染マウスからのリステリアの成功クリアランスは、宿主の免疫応答6の適切な発現に依存する。近交系マウス系統7,8の間で感度の広い範囲があります。一般に、 リステリア感染症にかかりやすくなることでマウスは抵抗性マウスに比べて増加細菌負荷および/ ​​または遅延クリアランスを示す、細菌の増殖を制御するために以下のことができます。連鎖解析とノックアウトマウス系統を含む遺伝学的研究は、配列の変化がリステリア感染6,8-14する宿主応答に影響を与えるための種々の遺伝子を同定した。異なるマウス系統間に感染動態の定量と比較すると、したがって、 リステリア菌に対する免疫応答に寄与する宿主遺伝要因を同定するための重要な方法です。別のリステリア菌に対する宿主応答の比較はまた、抗生物質療法やワクチンの設計のための潜在的なターゲットとして働くことができる細菌の病原性因子を同定するための効果的な方法です。 ここでは、細菌負荷（コロニー形成単位[CFU]あたりの組織）を測定し、 リステリア感染マウスにおける免疫応答のFACS分析のために肝臓や脾臓の単細胞懸濁液を調製するための簡単な方法を説明します。このメソッドは、最初の小説のマウス系統におけるリステリア感染の特性だけでなく、 リステリアに感染した別のマウスの系統間の免疫応答の比較のために特に有用である。私たちは、 リステリア菌を使用し、血液寒天培地上で培養したときに、ひずみ15 EGDβ-溶血1（図1）による各コロニーの周り特性ハローゾーンを示す。細菌負荷と免疫応答は、以下に記載されているプロトコルを使用してFACS解析のための血液寒天プレートと準備組織の細胞懸濁液での培養組織ホモジネートによる感染後の任意の時間の時点で決定することができます。我々は、免疫不全または妊娠している個人がリステリア菌処理ではないことに注意してだ、と仕事が始まる前に、関連する制度的なバイオセーフティ委員会及び動物施設の管理に相談する必要があります。

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

リステリア菌は、マウスの細胞内細菌に対する免疫応答と遺伝的感受性を研究するためのモデル生物です。このメソッドは、細菌の負荷を測定し、 リステリア感染による免疫細胞の変化を決定するためにFACS解析forマウスからの肝臓と脾臓の単細胞懸濁液を生成するものを可能にします。

に感染したマウスにおける細菌負荷と免疫応答の測定リステリア菌</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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Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

ジカ ウイルス特異 Immunoprivileged 臓器感染 Ifnar1-/-マウスの T 細胞応答の評価

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

リステリア菌脳の感染症

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