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

Die Autoren bedanken sich bei Anna Walduck, Christina Cheers, Stuart Berzins, Dale Godfrey, Yifan Zhan und Jonathan Wilksch für Beratung und Reagenzien zu danken. Diese Arbeit wurde von der Juvenile Diabetes Research Foundation (1-2008-602) und die Australian National Health and Medical Research Council (575552) gefördert. NW wird von einem Australian Postgraduate Award unterstützt. OW wird von einer RD Wright Fellowship von der Australian National Health Medical Research Council unterstützt.

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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. 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Keine Interessenskonflikte erklärt.

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> Name des Reagenzes </th><th> Firma </th><th> Katalog-Nummer </th><th> Kommentare </th></tr><tr><td> Pferd Blut-Agar-Basis No.2 </td><td> Oxoid </td><td> CM0271 </td><td> Vorbereitung der Agar-Basis für HBA-Platten </td></tr><tr><td> Pferd Blut (defibriniertes) </td><td> Oxoid </td><td> HB1000 </td><td> Fügen Sie 5-10% auf Agar-Basis </td></tr><tr><td> Brain Heart Infusion (BHI) Brühe (10 mL) </td><td> Oxoid </td><td> TM456 </td><td></td></tr><tr><td> Brain Heart Infusion dehydrierte Medien </td><td> Oxoid </td><td> CM1135 </td><td></td></tr><tr><td> 96-well Flachboden Platte </td><td> BD Biosciences </td><td> 353072 </td><td></td></tr><tr><td> 70 um Zellsieb </td><td> BD Biosciences </td><td> 352350 </td><td></td></tr><tr><td> Kleine Petrischale </td><td> BD Biosciences </td><td> 351007 </td><td></td></tr><tr><td> Stomacher 80 Biomaster Lab-System </td><td> Seward </td><td></td><td></td></tr><tr><td> Plastic Stomacherbeuteln </td><td> Sarstedt </td><td> 86 9924 530 </td><td></td></tr><tr><td> Rinderserumalbumin </td><td> Sigma </td><td> A3912 </td><td></td></tr><tr><td> FACS-Puffer </td><td></td><td></td><td> 0,1% (w / v) Rinderserumalbumin in PBS </td></tr><tr><td> Isotonischen Percoll (33,75% Percoll in PBS) </td><td> GE Healthcare </td><td> 17-0891-01 </td><td> 33.75mL Percoll, 3.75mL 10x PBS, macht 62.5mL 1x PBS 100ml isotonischer Percoll </td></tr><tr><td> TAC Buffer </td><td></td><td></td><td> 17mm Tris, 140mm Ammoniumchlorid in destilliertem Wasser </td></tr><tr><td> FCS / EDTA-Puffer </td><td></td><td></td><td> Fötalem Kälberserum mit 10mm EDTA </td></tr><tr><td> FACS / EDTA-Puffer </td><td></td><td></td><td> FACS-Puffer + 5 mM EDTA </td></tr><tr><td> Trypanblau </td><td> Sigma </td><td> T6146 </td><td> 0,4% (w / v) in PBS, sterilfiltriert </td></tr><tr><td> 2 ml-Kryoröhrchen </td><td> Greiner Bio One </td><td> 121263 </td><td></td></tr><tr><td> 27 gauge/1mL Insulinspritze </td><td> Terumo Medical Products </td><td> SS10M2713 </td><td></td></tr><tr><td> Nadel </td><td> Terumo Medical Products </td><td> NN-2516R (25G 5/8in) NN-2613R (26G 1/2in) </td><td></td></tr><tr><td> Spritze </td><td> Terumo Medical Produkte </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 (Listerien) ist ein Gram-positive fakultativ intrazellulären Erregers 1. Maus-Studien typischerweise intravenöser Injektion von Listeria, was zu einer systemischen Infektion 2. Nach der Injektion Listeria schnell verbreitet, die Milz und Leber durch die Aufnahme durch CD8α + dendritischen Zellen und Kupffer-Zellen 3,4. Sobald phagozytiert, ermöglichen verschiedene bakterielle Proteine ​​Listeria dem Phagosom entkommen, überleben im Cytosol und infizieren benachbarte Zellen 5. In den ersten drei Tagen der Infektion, vermitteln verschiedene angeborene Immunsystem-Zellen (zB Monozyten, Neutrophilen, NK-Zellen, dendritische Zellen) bakterizide Mechanismen, Listeria Verbreitung zu minimieren. CD8 + T-Zellen werden anschließend rekrutiert und für den eventuellen Clearance von Listerien aus dem Host, in der Regel innerhalb von 10 Tagen der Infektion 6. Erfolgreiche Clearance von Listerien aus infizierten Mäusen hängt von der entsprechenden Auftreten von Host Immunantwort 6. Es gibt ein breites Spektrum an Empfindlichkeiten unter Inzucht-Mausstämmen 7,8. Generell sind Mäuse mit einer erhöhten Anfälligkeit für Listeria Infektion weniger in der Lage, bakterielle Vermehrung steuern, zeigen erhöhte bakterielle Belastung und / oder verzögerte Clearance im Vergleich zu resistenten Mäusen. Genetische Studien, einschließlich Kopplungsanalyse und Knockout-Maus-Stämme wurden verschiedene Gene für die Sequenzvariation betrifft Host Reaktionen auf Listeria Infektion 6,8-14 identifiziert. Ermittlung und Vergleich der Infektion Kinetik zwischen verschiedenen Mausstämmen ist daher eine wichtige Methode zur Identifizierung von Host-genetischen Faktoren, die die Immunantwort gegen Listeria beitragen. Vergleich der Host-Reaktionen auf verschiedene Listeria-Stämme ist auch ein effektiver Weg zur bakteriellen Virulenzfaktoren, die als potenzielle Ziele für Antibiotika-Therapie oder Impfstoff-Design dienen könnten, zu erkennen. Wir beschreiben hier eine einfache Methode zur Messung der Keimbelastung (Kolonie bildende Einheiten [KBE] pro Gewebe) und die Vorbereitung Single-Cell-Suspensionen der Leber und der Milz für FACS-Analyse von Immunantworten in Listeria-infizierten Mäusen. Diese Methode ist besonders nützlich für die erstmalige Charakterisierung von Listeria-Infektionen bei neuartigen Maus-Stämme, sowie Vergleich der Immunantworten zwischen verschiedenen Mausstämmen mit Listeria infiziert. Wir nutzen die Listeria monocytogenes EGD-Stamm 15, die, wenn sie auf Blutagar kultiviert, weist eine charakteristische halo-Zone um jede Kolonie durch β-Hämolyse 1 (Abbildung 1). Bakterielle Belastung und Immunreaktionen können jederzeit-Punkt nach der Infektion durch Kultivierung Gewebehomogenat auf Blutagarplatten und Vorbereitung Gewebe Zellsuspensionen für FACS-Analyse unter Verwendung der Protokolle im Folgenden beschrieben wird. Wir möchte darauf hinweisen, dass Personen, die immungeschwächte oder schwanger sind, sollten nicht mit Listeria, und die einschlägigen institutionellen Kommission für Biosicherheit und Tier Facility Management sollte konsultiert werden, bevor die Arbeit beginnt werden.

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 ist ein Modellorganismus für die Untersuchung Immunreaktionen und die genetische Anfälligkeit für Bakterien bei Mäusen intrazellulär. Diese Methode ermöglicht ein bis bakterielle Belastung zu messen und generieren Single-Cell-Suspensionen der Leber und Milz von Mäusen für FACS-Analyse auf Veränderungen in Immunzellen durch Listeria-Infektion zu bestimmen.

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