We thank Sylvie Lesage and all lab members for critical reading of the protocol. This work was supported by a grant (MOP-77545) from the Canadian Institutes of Health Research (CIHR). Nathalie Labrecque is supported by a FRSQ Senior Scholarship and M&eacute;lissa Mathieu received a NSERC Alexander Graham Bell Canada Graduate Scholarship.

1. Preparation Before The Surgery
Mice were treated in accordance to the Canadian Council on Animal Care guidelines.
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	<li>Anesthetise mice by injection of ketamine/xylazine (150/300 mg/kg, i.p.) or by inhalation of isoflurane (2%, 1L oxygen). If available, isoflurane should be used as it allows for a better control of anaesthesia time and depth. Also, oxygen is administered at the same time which supports the physiological functions of the animal. Moreover, isoflurane is directly elimin...

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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=Complete+but+curtailed+T-cell+response+to+very+low-affinity+antigen.">Complete but curtailed T-cell response to very low-affinity antigen.</a> Nature. 458, 211-214 (2009).</li><li>Vezys, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Memory+CD8+T-cell+compartment+grows+in+size+with+immunological+experience.">Memory CD8 T-cell compartment grows in size with immunological experience.</a> Nature. 457, 196-199 (2009).</li><li>Zhou, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+and+persistence+of+memory+CD8(+)+T+cells+depend+on+T+cell+factor+1.">Differentiation and persistence of memory CD8(+) T cells depend on T cell factor 1.</a> Immunity. 33, 229-240 (2010).</li><li>Wirth, T. 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We have described a protocol of LN surgery which can apply to many experimental systems. Although very useful to follow immune responses or immune cell homeostasis, this technique has a few limitations. First, only four LNs can be harvested. Second, the immune response studied must be systemic or occur in the superficial inguinal or brachial (skin-draining) LNs. Finally, the number of cells harvested is influenced by the quality of the LN removal, a technical limitation which may contribute to experimental variability wh...

<table border="1">
<tbody>
 <tr>
 <td>Reagent Name</td>
 <td>Company</td>
 <td>Catalog number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Graefe Fine Pattern Premium Forceps </td>
 <td>Harvard Apparatus</td>
 <td>52-2144</td>
 <td>Strongly Curved, 10 cm (4 in), 0.8 mm tip</td>
 </tr>
 <tr>
 <td>Michel Clip Applying and Removing Forceps</td>
 <td>Harvard Apparatus</td>
 <td>52-3779</td>
 <td>12.5 cm (5 in) </td>
 </tr>
 <tr>
 <td>Michel Clip 100</td>
 <td>Harvard Apparatus</td>
 <td>52-3746</td>
 <td>7.5-1.75 mm</td>
 </tr>
 <tr>
 <td>Temgesic (Buprenorphine hydrochloride)</td>
 <td>Schering-Plough</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Vetalar (Ketamine hydrochloride)</td>
 <td>Boniche Animal Healthcare </td>
 <td>&nbsp;</td>
 <td>DIN: 01989529</td>
 </tr>
 <tr>
 <td>Rompun (Xylazine)</td>
 <td>Bayer</td>
 <td>&nbsp;</td>
 <td>DIN: 02169592</td>
 </tr>
 <tr>
 <td>Isoflurane</td>
 <td>ABBOTT</td>
 <td>&nbsp;</td>
 <td>DIN: 02032384</td>
 </tr>
 <tr>
 <td>Hypotears eye ointment</td>
 <td>Novartis Ophtalmics</td>
 <td>&nbsp;</td>
 <td>DIN: 02133288</td>
 </tr>
 <tr>
 <td>Baxedin (Clorhexidine gluconate 2% in isopropyl alcohol 70%)</td>
 <td>Omega</td>
 <td>L0000017</td>
 <td>DIN: 02251477</td>
 </tr>
 </tbody>
</table>

murine superficial lymph node surgery

In the field of immunology, to understand the progression of an immune response against a vaccine, an infection or a tumour, the response is often followed over time. Similarly, the study of lymphocyte homeostasis requires time course experiments. Performing these studies within the same mouse is ideal to reduce the experimental variability as well as the number of mice used. Blood withdrawal allows performance of time course experiments, but it only gives information about circulating lymphocytes and provides a limited number of cells1-4. Since lymphocytes circulating through the body and residing in the lymph nodes have different properties, it is important to examine both locations. The sequential removal of lymph nodes by surgery provides a unique opportunity to follow an immune response or immune cell expansion in the same mouse over time. Furthermore, this technique yields between 1-2x106 cells per lymph node which is sufficient to perform phenotypic characterization and/or functional assays. Sequential lymph node surgery or lymphadenectomy has been successfully used by us and others5-11. Here, we describe how the brachial and inguinal lymph nodes can be removed by making a small incision in the skin of an anesthetised mouse. Since the surgery is superficial and done rapidly, the mouse recovers very quickly, heals well and does not experience excessive pain. Every second day, it is possible to harvest one or two lymph nodes allowing for time course experiments. This technique is thus suitable to study the characteristics of lymph node-residing lymphocytes over time. This approach is suitable to various experimental designs and we believe that many laboratories would benefit from performing sequential lymph node surgeries.

Watch this Scientific Journal Video about Murine Superficial Lymph Node Surgery at JoVE.com

Murine Superficial Lymph Node Surgery

To follow the progression of an immune response over time within the same mouse, lymph nodes can be sequentially removed by surgery. Here, we describe how this technique can be performed.

In the field of immunology, to understand the progression of an immune response against a vaccine, an infection or a tumour, the response is often ...

Research

JoVE Journal

Immunology and Infection

41.8K visualizações.  Maisonneuve-Rosemont Hospital Research Center. Para seguir a progressão de uma resposta imune ao longo do tempo dentro do mesmo rato, gânglios linfáticos pode ser sequencialmente removido por cirurgia. Aqui, nós descrevemos como esta técnica pode ser realizada.