Gli autori desiderano ringraziare Anna Walduck, Christina Cheers, Stuart Berzins, Dale Godfrey, Yifan Zhan e Jonathan Wilksch per consigli e reagenti. Questo lavoro è stato finanziato dalla Juvenile Diabetes Research Foundation (1-2008-602) e la salute Australian National and Medical Research Council (575.552). NO è supportato da un premio post-laurea australiano. OW è supportato da una Wright RD Fellowship della Australian National Health Medical Research Council.

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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. 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Nessun conflitto di interessi dichiarati.

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> Nome del reagente </th><th> Azienda </th><th> Numero di catalogo </th><th> Commenti </th></tr><tr><td> Cavallo agar sangue di base n.2 </td><td> Oxoid </td><td> CM0271 </td><td> Preparazione di base per piastre di agar HBA </td></tr><tr><td> Cavallo sangue (defibrinato) </td><td> Oxoid </td><td> HB1000 </td><td> Aggiungi 5-10% a base di agar </td></tr><tr><td> Cervello infusione cuore (BHI) brodo (10 ml) </td><td> Oxoid </td><td> TM456 </td><td></td></tr><tr><td> Cervello cuore dei media infusione disidratati </td><td> Oxoid </td><td> CM1135 </td><td></td></tr><tr><td> 96 pozzetti fondo piatto </td><td> BD Biosciences </td><td> 353072 </td><td></td></tr><tr><td> 70 micron filtro cellulare </td><td> BD Biosciences </td><td> 352350 </td><td></td></tr><tr><td> Piccola scatola di Petri </td><td> BD Biosciences </td><td> 351007 </td><td></td></tr><tr><td> Stomacher 80 BIOMASTER laboratorio di sistema </td><td> Seward </td><td></td><td></td></tr><tr><td> Stomacher sacchetti di plastica </td><td> Sarstedt </td><td> 86 9924 530 </td><td></td></tr><tr><td> Albumina di siero bovino </td><td> Sigma </td><td> A3912 </td><td></td></tr><tr><td> FACS tampone </td><td></td><td></td><td> 0,1% (w / v) di BSA in PBS </td></tr><tr><td> Isotonica Percoll (33,75 Percoll% in PBS) </td><td> GE Healthcare </td><td> 17-0891-01 </td><td> 33.75mL Percoll, 3.75mL 10x PBS, 62.5mL 1x PBS fa 100 ml Percoll isotonico </td></tr><tr><td> TAC Buffer </td><td></td><td></td><td> 17mm Tris, cloruro di ammonio 140mm in acqua distillata </td></tr><tr><td> FCS / EDTA buffer di </td><td></td><td></td><td> Siero di vitello fetale con 10mM EDTA </td></tr><tr><td> FACS / EDTA buffer di </td><td></td><td></td><td> FACS tampone + 5mM EDTA </td></tr><tr><td> Blu tripano </td><td> Sigma </td><td> T6146 </td><td> 0,4% (w / v) in PBS, filtro sterilizzato </td></tr><tr><td> 2 ml provetta Microbank ™ </td><td> Greiner Bio One </td><td> 121263 </td><td></td></tr><tr><td> 27 siringa da insulina gauge/1mL </td><td> Terumo Medical Products </td><td> SS10M2713 </td><td></td></tr><tr><td> Ago </td><td> Terumo Medical Products </td><td> NN-2516R (25G 5/8in) NN-2613R (26G 1/2in) </td><td></td></tr><tr><td> Siringa </td><td> Terumo Medical Products </td><td> SS-01T (1 ml), SS-053 (5 ml), 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) è un batterio Gram-positivo patogeno intracellulare facoltativo 1. Studi sul topo tipicamente impiegano iniezione endovenosa di Listeria, che si traduce in infezione sistemica 2. Dopo l&#39;iniezione, Listeria diffonde rapidamente alla milza e al fegato a causa di assorbimento da parte CD8α + cellule dendritiche e le cellule di Kupffer 3,4. Una volta fagocitati, varie proteine ​​batteriche Listeria consentono di sfuggire alla phagosome, sopravvivono all&#39;interno del citosol, e infettare le cellule vicine 5. Durante i primi tre giorni di infezione, le cellule differenti immunitario innato (es. monociti, neutrofili, cellule NK, cellule dendritiche) mediare i meccanismi battericida che riducono al minimo la proliferazione di Listeria. Cellule T CD8 + vengono successivamente assunti e responsabile per la liquidazione finale di Listeria dal host, in genere entro 10 giorni di infezione 6. Distanza di successo di Listeria da topi infettati dipende l&#39;insorgenza appropriato di risposta immunitaria 6. Vi è un&#39;ampia gamma di sensibilità tra i ceppi di topi inbred 7,8. In genere, i topi con una maggiore suscettibilità alle infezioni da Listeria sono meno in grado di controllare la proliferazione batterica, dimostrando una maggiore carica batterica e / o clearance ritardata rispetto ai topi resistenti. Studi genetici, comprese le analisi di linkage e ceppi di topi knockout, hanno identificato i geni diversi per i quali variazione di sequenza colpisce risposte serie alle infezioni da Listeria 6,8-14. Determinazione e confronto delle cinetiche di infezione tra i diversi ceppi di topi è quindi un importante metodo per identificare i fattori dell&#39;ospite genetici che contribuiscono alla risposta immunitaria contro Listeria. Confronto di risposte serie a diversi ceppi di Listeria è anche un modo efficace per identificare fattori di virulenza dei batteri che possono servire come potenziali bersagli per la terapia antibiotica o la progettazione di vaccini. Descriviamo qui un metodo semplice per la misurazione della carica batterica (unità formanti colonia [CFU] tessuto per persona) e la preparazione a cella singola sospensioni del fegato e della milza per l&#39;analisi FACS delle risposte immunitarie in topi infettati con Listeria. Questo metodo è particolarmente utile per la caratterizzazione iniziale di infezione da Listeria in ceppi di topi romanzo, così come il confronto delle risposte immunitarie tra diversi ceppi di topi infettati con Listeria. Usiamo il Listeria monocytogenes EGD ceppo 15 che, se coltivati ​​su agar sangue, presenta una zona caratteristica alone intorno ad ogni colonia a causa di β-emolisi 1 (Figura 1). Carica batterica e le risposte immunitarie possono essere determinati in qualsiasi momento-point dopo l&#39;infezione da tessuto omogenato coltura su piastre di agar sangue e la preparazione delle sospensioni di cellule di tessuto per l&#39;analisi FACS utilizzando i protocolli descritti di seguito. Si segnala che gli individui immunocompromessi o in stato di gravidanza non devono maneggiare Listeria, e il comitato di biosicurezza istituzionali competenti e facility management animale deve essere consultato prima dei lavori.

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 è un organismo modello per studiare le risposte immunitarie e la suscettibilità genetica a batteri intracellulari nei topi. Questo metodo permette di misurare la carica batterica e di generare monocellulari sospensioni del fegato e della milza di topi per l&#39;analisi FACS per determinare cambiamenti nelle cellule del sistema immunitario dovuta a infezione da Listeria.

Misurazione della carica batterica e Risposte immunitarie in topi infettati con Listeria 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

30.8K Views.  The University of Melbourne. Listeria monocytogenes è un organismo modello per studiare le risposte immunitarie e la suscettibilità genetica a batteri intracellulari nei topi. Questo metodo permette di misurare la carica batterica e di generare monocellulari sospensioni del fegato e della milza di topi per l'analisi FACS per determinare cambiamenti nelle cellule del sistema immunitario dovuta a infezione da Listeria.