Name: murine superficial lymph node surgery
Uploaded: 2012-05-21T12:00:00
Description: Watch this Scientific Journal Video about Murine Superficial Lymph Node Surgery at JoVE.com

Vi takker Sylvie Lesage og alle lab medlemmer for kritisk lesing av protokollen. Dette arbeidet ble støttet av en bevilgning (MOP-77545) fra den kanadiske Institutes of Health Research (CIHR). Nathalie Labrecque støttes av en FRSQ Senior stipend og Melissa Mathieu fikk en NSERC Alexander Graham Bell Canada Graduate Scholarship.

1. Forberedelser før operasjonen Musene ble behandlet i henhold til den kanadiske rådet på Animal Care retningslinjer. <ol><li> Anesthetise mus ved injeksjon av ketamin / xylazin (150/300 mg / kg, ip) eller ved innånding av isofluran (2%, 1L oksygen). Hvis tilgjengelig, bør isofluran brukes som det gir en bedre kontroll av anestesi tid og dybde. Dessuten er oksygen administreres samtidig som støtter fysiologiske funksjoner i dyret. Videre er isofluran direkte eliminert av lungene og ...

<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=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. C., Badovinac, V. P., Zhao, L., Dailey, M. O., Harty, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+central+memory+CD8+T+cells+is+independent+of+CD62L-mediated+trafficking+to+lymph+nodes.">Differentiation of central memory CD8 T cells is independent of CD62L-mediated trafficking to lymph nodes.</a> J. Immunol. 182, 6195-6206 (2009).</li><li>Rooke, R., Waltzinger, C., Benoist, C., Mathis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+complementation+of+MHC+class+II+deficiency+by+intrathymic+delivery+of+recombinant+adenoviruses.">Targeted complementation of MHC class II deficiency by intrathymic delivery of recombinant adenoviruses.</a> Immunity. 7, 123-134 (1997).</li><li>Witherden, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetracycline-controllable+selection+of+CD4(+)+T+cells:+half-life+and+survival+signals+in+the+absence+of+major+histocompatibility+complex+class+II+molecules.">Tetracycline-controllable selection of CD4(+) T cells: half-life and survival signals in the absence of major histocompatibility complex class II molecules.</a> J. Exp. 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Vi har beskrevet en protokoll av LN kirurgi som kan gjelde for mange eksperimentelle systemer. Selv om svært nyttig å følge immunreaksjoner eller immun celle homeostase, har denne teknikken noen begrensninger. Først kan kun fire LNS høstes. Andre må immunresponsen studerte være systemisk eller forekommer i de overfladiske lysken eller brakial (hud-drenering) LNS. Endelig er antall celler høstet påvirket av kvaliteten på LN fjerning, en teknisk begrensning som kan bidra til eksperimentell variasjon når absolut...

<table border="1"><tbody><tr><td> Reagens Navn </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer </td></tr><tr><td> Graefe fine mønster Premium tang </td><td> Harvard Apparatus </td><td> 52-2144 </td><td> Sterkt buet, 10 cm (4 tommer), 0,8 mm tip </td></tr><tr><td> Michel Clip påfører og fjerner tang </td><td> Harvard Apparatus </td><td> 52-3779 </td><td> 12,5 cm (5 cm) </td></tr><tr><td> Michel Clip 100 </td><td> Harvard Apparatus </td><td> 52-3746 </td><td> 7,5 til 1,75 mm </td></tr><tr><td> Temgesic (Buprenorfin hydroklorid) </td><td> Schering-Plough </td><td></td><td></td></tr><tr><td> Vetalar (Ketamin hydroklorid) </td><td> Boniche Animal Healthcare </td><td></td><td> DIN: 01989529 </td></tr><tr><td> Rompun (xylazin) </td><td> Bayer </td> &lt;td&gt; </td><td> DIN: 02169592 </td></tr><tr><td> Isofluran </td><td> ABBOTT </td><td></td><td> DIN: 02032384 </td></tr><tr><td> Hypotears øyesalve </td><td> Novartis Ophtalmics </td><td></td><td> DIN: 02133288 </td></tr><tr><td> Baxedin (Clorhexidine gluconate 2% i isopropylalkohol 70%) </td><td> Omega </td><td> L0000017 </td><td> DIN: 02251477 </td></tr></tbody></table>

murine superficial lymph node surgery

Innen immunologi, for å forstå utviklingen av en immunrespons mot en vaksine, en infeksjon eller en svulst, er responsen ofte fulgt over tid. Tilsvarende krever studiet av lymfocytt homeostase tidsforløpet eksperimenter. Utføre disse studiene innenfor samme musen er ideelt å redusere den eksperimentelle variabilitet samt antall mus brukes. Blood uttak tillater utførelse av tidsforløpet eksperimenter, men det gir bare informasjon om sirkulerende lymfocytter og gir et begrenset antall celler 1-4. Siden lymfocytter sirkulerer gjennom kroppen og bosatt i lymfeknutene har forskjellige egenskaper, er det viktig å undersøke begge steder. Den sekvensielle fjerning av lymfeknuter ved kirurgi gir en unik mulighet til å følge en immunrespons eller immun celle ekspansjon i samme musen over tid. Videre gir denne teknikken mellom 1-2x10 6 celler per lymfeknute som er tilstrekkelig til å perform fenotypisk karakterisering og / eller funksjonelle analyser. Sekvensiell lymfeknute kirurgi eller lymphadenectomy har blitt brukt av oss og andre 5-11. Her beskriver vi hvordan brakiale og inguinal lymfeknuter kan fjernes ved å lage et lite snitt i huden av en bedøvet mus. Siden operasjonen er overfladisk og gjort raskt, gjenoppretter musa veldig raskt, helbreder godt og ikke opplever overdreven smerte. Annenhver dag, er det mulig å høste en eller to lymfeknuter som åpner for tidsforløpet eksperimenter. Denne teknikken er derfor egnet til å studere egenskapene til lymfeknute-bosatt lymfocytter over tid. Denne tilnærmingen er egnet til ulike eksperimentelle design, og vi tror at mange laboratorier ville ha nytte av å utføre sekvensielle lymfeknute surgeries.

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

Murine Superficial Lymph Node Surgery

Å følge utviklingen av en immunrespons over tid innenfor samme mus, kan lymfeknuter være sekvensielt fjernes ved operasjon. Her beskriver vi hvordan denne teknikken kan utføres.

Murint Overflatisk lymfeknute 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 ...

Research

JoVE Journal

Immunology and Infection

Intravital Microscopy of the Inguinal Lymph Node

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune ...

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration ...

Intravital Imaging of the Mouse Popliteal Lymph Node

Intravital Imaging av Mouse popliteal lymfeknute

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign ...

Generation of Lymph Node-fat Pad Chimeras for the Study of Lymph Node Stromal Cell Origin

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the ...

Isolation of Murine Lymph Node Stromal Cells

Isolering av Murint Lymph Node stromale celler

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and ...

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

Myelogen Cell Isolasjon fra Mouse Hud og Drenering Lymph Node Etter Intradermal Immunisering med levende svekket<em&gt; Plasmodium</em&gt; Sporozoitter

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly ...

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with Mycobacterium bovis Bacille Calmette-GuÃƒÆ’Ã†'Ãƒâ€šÃ‚Â©rin

En CFSE basert analyse for å studere migrasjon av murine Skin dendrittiske celler i Tømming lymfeknuter Under infeksjon med<em&gt; Mycobacterium bovis</em&gt; Bacille Calmette-Guérin

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph ...

Murine Full-thickness Skin Transplantation

Murine full-tykkelse hudtransplantasjon

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited ...

Intracellular Staining and Flow Cytometry to Identify Lymphocyte Subsets within Murine Aorta, Kidney and Lymph Nodes in a Model of Hypertension

Intracellulær Farging og flowcytometri for å identifisere Lymfocytt Delsett i Murine Aorta, Nyre og lymfeknuter i en Model of Hypertension

It is now well known that T lymphocytes play a critical role in the development of several cardiovascular diseases1,2,3,4,5. For example, studies from our ...

Collection and Processing of Lymph Nodes from Large Animals for RNA Analysis: Preparing for Lymph Node Transcriptomic Studies of Large Animal Species

Innsamling og behandling av Lymph noder fra store dyr for RNA analyse: forbereder lymfeknute Transcriptomic studier av store dyrearter

Large animals (both livestock and wildlife) serve as important reservoirs of zoonotic pathogens, including Brucella, Mycobacterium bovis, Salmonella, and ...