We are grateful to the personnel of the Biomedical Services Unit of the University of Birmingham for taking care of our animal colonies. This work was supported by the EU FP7 integrated project INFLACARE to JC.

Mice were bred and maintained under SPF conditions in the Biomedical Service Unit at the University of Birmingham according to UK Home Office and local ethics committee regulations. All procedures described in this protocol are covered under a Project License approved by both local ethics committee and the Home Office.
1. Isolation of Newborn LNs
<ol>
	<li>Sacrifice the newborn mice by cervical dislocation.</li>
	<li>Section the head, and open the body with scissors from the top of the thoracic region to the bottom of the abdominal region and remove carefully all the viscera (heart, lungs, liver, intestine, kidney, bladder) from the abdominal cavity.</li>
	<li>Transfer the body into a 90 mm Petri dish containing RF10 media (RPMI1640 media supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 mg/ml streptomycin and 2 mM L-glutamine). From this step onwards, the tissues will be kept under sterile conditions and handled in a tissue culture hood.</li>
	<li>Transfer the body into a new 90 mm Petri dish containing RF10.</li>
	<li>Under the dissecting microscope, detach carefully the peritoneum from the skin in the inguinal region. The inguinal LN is situated at the intersection of three blood vessels in the fat pad.</li>
	<li>Remove carefully the LN. Make sure that all adipose tissue is removed. Transfer the LN into a 50 mm Petri dish containing RF10 media. Keep the tissues on ice while continuing with the procedure.</li>
</ol>
2. Isolation of E18.5 Fat Pads
<ol>
	<li>To generate mouse embryos at day 18.5 of gestation (E18.5), set up timed pregnancies by placing a female and a male mouse per cage overnight. Separate the mice in the morning and check for vaginal plugs (gestational day E0.5).
	<ol>
		<li>Euthanize the plugged females at day 18.5 of pregnancy by cervical dislocation.</li>
		<li>Remove the embryos and placed them in a 90 mm Petri Dish containing RF10.</li>
	</ol>
	</li>
	<li>From this step onwards, tissues should be handled using sterile technique in the tissue culture hood. Under the dissecting microscope, section the head, open the embryos with scissors from the thoracic region to the bottom of the abdominal region and remove carefully all the viscera (heart, lungs, liver, intestine, kidney, bladder) from the body cavity.</li>
	<li>Clean the body from all remaining visceral tissues and wash it in PBS to eliminate all traces of blood that could make the dissection more difficult.</li>
	<li>Transfer the clean bodies into a fresh 90 mm Petri dish containing RF10.</li>
	<li>Carefully detach the peritoneum from the skin in the inguinal region. The inguinal LN is situated at the intersection of three blood vessels in the fat pad.</li>
	<li>Carefully remove the LN and discard it. It is very important to completely remove the LN to ensure that the fat pad used for the chimeras is not contaminated by any lymphoid stromal cells.</li>
	<li>Remove the inguinal fat pad and transfer it into a 50 mm Petri dish containing RF10 media. Keep the tissues on ice while continuing with the procedure.</li>
</ol>
3. Generation of the LN-fat Pad Chimera
<ol>
	<li>Prepare the in vitro organ culture system15.
	<ol>
		<li>Sponge and filters preparation: This can be done in advance and sponges and filters stored sterile in a Petri dish in the culture hood.
		<ol>
			<li>Cut the Vulkan Underwrap in 1-1.5 cm2 pieces</li>
			<li>Boil the sponges for 2 hr in distilled water.</li>
			<li>Let the sponges dry for a couple of hours in the cell culture hood.</li>
			<li>Boil the filters for 20 min.</li>
			<li>Let the filters dry for a couple of hours in the cell culture hood.</li>
		</ol>
		</li>
		<li>Prepare DMEM media complemented with 10% fetal bovine serum, 10 mM HEPES, 1x&#160;MEM Nonessential Amino Acid Solution, 50 mM 2-Mercaptoethanol, 100 U/ml penicillin, 100 mg/ml streptomycin and 2 mM L-Glutamine.</li>
		<li>Add 2 ml of media in a 50 mm Petri dish.</li>
		<li>Arrange one sponge at the bottom of the Petri dish. Wet both sides of the sponge by submerging them in the media.</li>
		<li>Place one filter on top of the sponge. The filter is positioned at the liquid-air interface.</li>
	</ol>
	</li>
	<li>Under the dissecting microscope, carefully reassociate one newborn LN with one embryonic fat pad.</li>
	<li>Place the LN-fat pad chimera on top of the filter.</li>
	<li>Transfer the Petri dish in a rectangular plastic box with a couple of holes on the lid and containing water at the bottom to ensure humidity.</li>
	<li>Seal the lid to the box with tape. Leave the two holes in the lid open.</li>
	<li>Transfer the box to a cell culture incubator at 37 &#176;C with 5% CO2. Allow the box to equilibrate for 2 hr, before sealing the holes in the lid with tape.</li>
	<li>Incubate the tissues for at least 2 days to allow the LN to attach to the fat pad and be transferable under the kidney capsule.</li>
</ol>
4. Transfer of the LN-fat Pad Chimera Under the Kidney Capsule of Host Mice
<ol>
	<li>Collect the LN-fat pad chimeras from the filter and transfer them under the kidney capsule of host mice. The kidney capsule transfer method has already been described 16.</li>
</ol>
5. Isolation of the Transferred LNs
<ol>
	<li>After 3-4 weeks under the kidney capsule, the LN-fat pads chimeras are ready to be harvested. Euthanize the host mice using approved guidance.</li>
	<li>Remove the kidney from the host mice.</li>
	<li>Under the dissecting microscope, isolate the LN-fat pad chimera and dissect out the LN.</li>
</ol>
6. Enzymatic Digestion of the Transferred LNs for Flow-Cytometric Analysis
<ol>
	<li>Make one incision in the LN with small scissors to allow the enzymes to penetrate the organ and facilitate the digestion.</li>
	<li>Place one LN in 1.5 ml Eppendorf containing 600 &#956;l of digestion buffer (RPMI 1% FCS, 2.5 mg/ml Collagenase D, 100 &#956;g/ml DNase I).</li>
	<li>Incubate for 30 min at 37 &#176;C on an agitating thermal block. Pipette up and down to help dissociation of tissues every 10 min.</li>
	<li>Add 6 &#956;l EDTA 0.5 M to the tube and pipette up and down to finish the dissociation of the tissues.</li>
	<li>Centrifuge for 5 min at 490 x g.</li>
	<li>Resuspend the cell pellet in the staining solution (PBS, 0.1% BSA, 5% rat serum and 5% mouse serum) containing the combination of primary antibodies.</li>
	<li>Incubate for 30 min on ice.</li>
	<li>Wash twice in PBS</li>
	<li>Incubate with secondary antibodies for 20 min if required.</li>
	<li>Wash 2x in PBS.</li>
	<li>Analyze on flow-cytometer.</li>
</ol>
7. Analysis of the Transferred LNs by Immunofluorescence
<ol>
	<li>Fixation for EYFP conservation during the freezing and cryosections process: Place the LN in a solution of PBS, 4% PFA and 10% sucrose and leave for 3-4 hr.</li>
	<li>Put a single small drop of cold O.C.T. compound on a small piece of aluminum foil. Avoid air bubbles. Carefully place the LN without any liquid in the middle of the drop.</li>
	<li>Freeze the organ by placing the aluminum foil on dry ice.</li>
	<li>Cut 8-10 &#956;m sections onto extra adhesives glass slides using a cryostat. Let the slides dry for 2 hr before storing them at -20 &#176;C.</li>
	<li>Staining procedures can vary according to primary antibodies used and can be optimized from the following protocol. Stain the sections with staining solution (PBS, 1% BSA) containing the combination of primary antibodies.</li>
	<li>Incubate for 1 hr at room temperature in a humidified chamber.</li>
	<li>Wash twice in PBS for 5 min</li>
	<li>Incubate with secondary antibodies for 1 hr.</li>
	<li>Wash twice in PBS for 5 min.</li>
	<li>Mount slides in mounting media containing DAPI.</li>
	<li>Capture images using a fluorescence or confocal microscope.</li>
</ol>

<ol>
	<li>Mueller, S. N., Germain, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal+cell+contributions+to+the+homeostasis+and+functionality+of+the+immune+system.">Stromal cell contributions to the homeostasis and functionality of the immune system.</a> Nat. Rev. Immunol. 9, 618-629 (2009).</li><li>Roozendaal, R., Mebius, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal+cell-immune+cell+interactions.">Stromal cell-immune cell interactions.</a> Annu. Rev. Immunol. 29, 23-43 (2011).</li><li>Turley, S. J., Fletcher, A. L., Elpek, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+stromal+and+haematopoietic+antigen-presenting+cells+that+reside+in+secondary+lymphoid+organs.">The stromal and haematopoietic antigen-presenting cells that reside in secondary lymphoid organs.</a> Nat. Rev. Immunol. 10, 813-825 (2010).</li><li>Katakai, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organizer-like+reticular+stromal+cell+layer+common+to+adult+secondary+lymphoid+organs.">Organizer-like reticular stromal cell layer common to adult secondary lymphoid organs.</a> J. Immunol. 181, 6189-6200 (2008).</li><li>Link, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblastic+reticular+cells+in+lymph+nodes+regulate+the+homeostasis+of+naive+T+cells.">Fibroblastic reticular cells in lymph nodes regulate the homeostasis of naive T cells.</a> Nat. Immunol. 8, 1255-1265 (2007).</li><li>Benezech, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ontogeny+of+stromal+organizer+cells+during+lymph+node+development.">Ontogeny of stromal organizer cells during lymph node development.</a> J. Immunol. 184, 4521-4530 (2010).</li><li>Cupedo, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presumptive+lymph+node+organizers+are+differentially+represented+in+developing+mesenteric+and+peripheral+nodes.">Presumptive lymph node organizers are differentially represented in developing mesenteric and peripheral nodes.</a> J. Immunol. 173, 2968-2975 (2004).</li><li>Avan de Pavert, S., Mebius, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+the+development+of+lymphoid+tissues.">New insights into the development of lymphoid tissues.</a> Nat. Rev. Immunol. 10, 664-674 (2010).</li><li>Vondenhoff, M. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LTbetaR+signaling+induces+cytokine+expression+and+up-regulates+lymphangiogenic+factors+in+lymph+node+anlagen.">LTbetaR signaling induces cytokine expression and up-regulates lymphangiogenic factors in lymph node anlagen.</a> J. Immunol. 182, 5439-5445 (2009).</li><li>Dougall, W. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RANK+is+essential+for+osteoclast+and+lymph+node+development.">RANK is essential for osteoclast and lymph node development.</a> Genes Dev. 13, 2412-2424 (1999).</li><li>Kim, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+peripheral+lymph+node+genesis+by+the+tumor+necrosis+factor+family+member+TRANCE.">Regulation of peripheral lymph node genesis by the tumor necrosis factor family member TRANCE.</a> J. Exp. Med. 192, 1467-1478 (2000).</li><li>Carragher, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+stroma-derived+defect+in+NF-kappaB2-/-+mice+causes+impaired+lymph+node+development+and+lymphocyte+recruitment.">A stroma-derived defect in NF-kappaB2-/- mice causes impaired lymph node development and lymphocyte recruitment.</a> J. Immunol. 173, 2271-2279 (2004).</li><li>White, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphotoxin+a-dependent+and+-independent+signals+regulate+stromal+organizer+cell+homeostasis+during+lymph+node+organogenesis.">Lymphotoxin a-dependent and -independent signals regulate stromal organizer cell homeostasis during lymph node organogenesis.</a> Blood. 110, 1950-1959 (2007).</li><li>Benezech, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphotoxin-beta+receptor+signaling+through+NF-kappaB2-RelB+pathway+reprograms+adipocyte+precursors+as+lymph+node+stromal+cells.">Lymphotoxin-beta receptor signaling through NF-kappaB2-RelB pathway reprograms adipocyte precursors as lymph node stromal cells.</a> Immunity. 37, 721-734 (2012).</li><li>Anderson, G., Jenkinson, E. J., Moore, N. C., Owen, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MHC+class+II-positive+epithelium+and+mesenchyme+cells+are+both+required+for+T-cell+development+in+the+thymus.">MHC class II-positive epithelium and mesenchyme cells are both required for T-cell development in the thymus.</a> Nature. 362, 70-73 (1993).</li><li>Szot, G. L., Koudria, P., Bluestone, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+pancreatic+islets+into+the+kidney+capsule+of+diabetic+mice.">Transplantation of pancreatic islets into the kidney capsule of diabetic mice.</a> J. Vis. Exp. (9), e404 (2007).</li><li>Krautler, N. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Follicular+dendritic+cells+emerge+from+ubiquitous+perivascular+precursors.">Follicular dendritic cells emerge from ubiquitous perivascular precursors.</a> Cell. 150, 194-206 (2012).</li></ol>

The authors have nothing to disclose.

In this article we presented a method to assay and quantify the contribution of adipose tissue progenitor cells to the developing LNs and two techniques that allow the analysis of their progeny. Dissection of embryonic fat pads and newborns LNs are delicate and require manual skills gained by a lot of practice prior to the generation of the actual LN-fat pad chimera. To control the quality of the dissections, flow-cytometric analysis can be performed on the fat pads and LNs. Embryonic fat pad preparation should be devoid of lymph nodes and should not contain any CD45+CD4+IL-7Ra+ lymphoid tissue inducer cells. Newborns LN preparations should be completely free of adipose tissue and contain a high proportion of CD45-CD31-ICAM-1highVCAM-1high lymphoid tissue organizer cells6,13-14. Standard sterile techniques for tissue culture and surgery should be followed to reduce the risk of contamination.	
	Engagement of the LT&beta;R on immature LN stromal cells induces their differentiation into fully mature LN stromal cells. Using this in vivo system, we previously demonstrated that fat pads derived from a mouse deficient for LT&beta;R are not able to sustain the growth of the LN. Therefore, this technique can be used with fat pads derived from different knock-out mouse models to assess the need for adipose tissue derived cells in the development of different LN stromal cell subsets. It is not known yet whether adipose tissue can give rise to all LN stromal cell populations. Combinations of LNs and fat pad of different ages could be used to determine the presence of progenitor cells at varying time points. This approach can also be combined with the use of recipient mice expressing another cell marker, such as RFP, to compare the requirement in local and distant progenitor cells for the LN growth.	
	During an immune response, the LN undergoes a dramatic expansion, including a marked remodeling and expansion of the stromal cell network. However, very little is known about the mechanisms regulating these processes. This in vivo system, could be used with adult adipose tissue and adults LNs in combination with a systemic immunization17 to assess the role of adipose tissue derived cells in the remodeling of the LN stroma during immune responses.

Lymph nodes (LNs) are key organs of the immune system situated at strategic sites in the body, along the lymphatic vasculature network. They enable filtration of antigens and pathogens and provide a site for antigen presentation to lymphocytes and induction of adaptive immune responses. The stroma, which forms the basic structure of the LN and orchestrates the movement of the different hematopoietic participants of the adaptive immune response, is central to the function of these organs. Different populations of stromal cells supply essential and specific cues for the movements, localization, survival, proliferation and maturation of the hematopoietic component of the immune system1-3. Adult LN stromal cells fall in three categories: the blood endothelial cells, the lymphatic endothelial cells and fibroblasts. These three categories encompass heterogeneous populations. The fibroblastic populations contain fibroblastic reticular cells (FRC), follicular dendritic cells (FDC), marginal reticular cells (MRC), while the fibroblasts forming the capsule, the medulla and other cells which are not yet identified1-5. The origins and the mechanisms governing the maturation of the different LN stromal cell populations are unclear and the absence of specific markers allowing the fate mapping of specific LN stromal cell populations rends their study particularly difficult. However, a full understanding of the ontogeny of LN stromal cells is necessary to the comprehension of adaptive immune responses, the mechanisms contributing to tolerance and is at the basis of the development of artificial LNs.
So far, the study of LN stromal cell origin and development has been mostly limited to the direct assessment of LN development in embryos and newborns in wild type mice and different knockout mouse strains6-9. These approaches are limited by the embryonic and perinatal lethality of some of the mouse strains carrying deletions in genes important for LN development. Moreover, some of the genes essential for lymphoid tissue development are also involved in a wide range of biological process as it is the case for RANK10-11 or NF-&#954;B212. To address these issues, isolation and transplantation of embryonic LNs under the kidney capsule of a host mouse have been performed12-13. This technique allows, for example, the transfer of genetically modified embryonic LNs in a wild type environment to assess organ development and the recruitment and organization of host cells. However, the growth of embryonic LN grafted under the kidney capsule of an adult host is impaired, thus limiting the use of this technique.
LNs and fat deposits are anatomically closely associated and they develop simultaneously during embryogenesis. We recently demonstrated that the association LN/adipose tissue plays a crucial role in the provision of stromal cell progenitors for the LNs. In particular, signaling through the LT&#946;R controls the fate of adipocyte precursor cells by blocking adipogenesis and instead promoting maturation towards a LN stromal cell phenotype14. Here we describe a method based on generation of LN-fat pad chimeras allowing the tracing of adipose tissue derived cells in the developing LN. This method will be useful to determine the contribution of adipose tissues to different LN stromal cell populations and combined with the use of tissues from genetically modified mouse strains, will allow a better understanding of the mechanisms controlling the differentiation of the different LN stromal cell subsets.

<table border="1">
	<tbody>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma</td>
			<td>M3148</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mm Sterilin Petri Dish</td>
			<td>Appleton Woods</td>
			<td>SC265</td>
			<td></td>
		</tr>
		<tr>
			<td>90 mm Sterilin Petri Dish</td>
			<td>Appleton Woods</td>
			<td>SC260</td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesive slides</td>
			<td>Surgipath</td>
			<td>00202</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD31 eFluor 450</td>
			<td>eBioscience</td>
			<td>48-0311</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD4 Alexa 647</td>
			<td>eBioscience</td>
			<td>51-0041</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD45 PerCP-Cy5.5</td>
			<td>eBioscience</td>
			<td>45-0451</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse IgM Rhodamine Red</td>
			<td>Stratech</td>
			<td>715-296-020-JIR</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse Podoplanin PE/Cy7</td>
			<td>Biolegend</td>
			<td>127411</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse Podoplanin purified</td>
			<td>eBioscience</td>
			<td>14-5381</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase D</td>
			<td>Roche</td>
			<td>11088858001</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM Medium</td>
			<td>Sigma</td>
			<td>D5671</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Sigma</td>
			<td>DN25-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps Inox Biologie</td>
			<td>FST</td>
			<td>11252-20</td>
			<td>Extra fine forceps for embryonic and newborn dissections</td>
		</tr>
		<tr>
			<td>EDTA solution 0.5 M</td>
			<td>Sigma</td>
			<td>E7889</td>
			<td></td>
		</tr>
		<tr>
			<td>ERTR-7</td>
			<td>Biogenesis</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Sigma</td>
			<td>F9665</td>
			<td></td>
		</tr>
		<tr>
			<td>Hardened fine iris scissors straight 11 cm</td>
			<td>FST</td>
			<td>14090-11</td>
			<td>Small scissors for dissection</td>
		</tr>
		<tr>
			<td>HEPES solution</td>
			<td>Sigma</td>
			<td>H0887</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopore Membrane Filters 0.8 &#956;m ATTP</td>
			<td>Millipore</td>
			<td>ATTPO 1300</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine solution</td>
			<td>Sigma</td>
			<td>G7513</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Nonessential Amino Acid Solution (100x)</td>
			<td>Sigma</td>
			<td>M7145</td>
			<td></td>
		</tr>
		<tr>
			<td>O.C.T. Compound</td>
			<td>Tissue-Tek</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Sigma</td>
			<td>P4458</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic box with lid</td>
			<td>Watkins and Doncaster</td>
			<td>E6052</td>
			<td>Sandwich box for in vitro organ culture. Drill two holes in the lid to allow gas exchange in the CO2 incubator.</td>
		</tr>
		<tr>
			<td>RPMI 1640 Medium</td>
			<td>Sigma</td>
			<td>R0883</td>
			<td></td>
		</tr>
		<tr>
			<td>Sponges Vulkan Underwrap</td>
			<td>Patterson Medical</td>
			<td>004383</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Leica</td>
			<td>LEICA MZ95</td>
			<td>Dissecting microscope with zoom</td>
		</tr>
		<tr>
			<td>Thermomixer</td>
			<td>Eppendorf</td>
			<td>5436</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectashield Mounting Medium with DAPI</td>
			<td>Vector Laboratories</td>
			<td>H-1200</td>
			<td></td>
		</tr>
	</tbody>
</table>

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 mechanisms governing its formation. Lymph nodes are always encapsulated in adipose tissue and we recently demonstrated the importance of this relation for the formation of lymph node stroma. Adipocyte precursor cells migrate into the lymph node during its development and upon engagement of the Lymphotoxin-b receptor switch off adipogenesis and differentiate into lymphoid stromal cells (B&#233;n&#233;zech&#160;et al.14). Based on the lymphoid stroma potential of adipose tissue, we present a method using a lymph node/fat pad chimera that allows the lineage tracing of lymph node stromal cell precursors. We show how to isolate newborn lymph nodes and EYFP+ embryonic adipose tissue and make a LN/ EYFP+ fat pad chimera. After transfer under the kidney capsule of a host mouse, the lymph node incorporates local adipose tissue precursor cells and finishes its formation. Progeny analysis of EYFP+ fat pad cells in the resulting lymph nodes can be performed by flow-cytometric analysis of enzymatically digested lymph nodes or by immunofluorescence analysis of lymph nodes cryosections. By using fat pads from different knockout mouse models, this method will provide an efficient way of analyzing the origin of the different lymph node stromal cell populations.

Three weeks after transplantation, the LN/fat pad chimera is recovered from the kidney. The chimera is now very similar to a normal LN in its own fat pad, and the LN is visible in the center of the adipose tissue (Figure 1). If the LN can&#39;t be identified, it is possible that it was lost during the transplantation on the kidney. When assessing the role of genes potentially important in LN development, the LNs recovered may remain very small and more difficult to find. This is the case when adipose tissue from LT&#946;R-/- mice is reassociated with newborn LNs14.
Careful isolation of the LN allows further analysis of the progeny of EYFP+ adipose derived cells. Cryosections and immunofluorescence analysis of the LN reveal that EYFP+ adipose derived cells migrate into the LN where they contribute to the Gp38+ERTR-7+ LN stromal cell network (Figure 2A). Flow-cytometric analysis confirms that an important fraction of LN stromal cells derives from local EYFP+ adipose tissue progenitor cells, contributing to 30% of the CD45-Gp38+CD31- fibroblastic fraction and 10% of the CD45-Gp38-CD31+ blood endothelial cell fraction (Figure 2B). Up to 80% of the CD45-Gp38+CD31- fibroblastic fraction can derived from adipose tissue precursor cells demonstrating the crucial role played by adipose tissue in sustaining the growth of the LN stroma.
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/50952/50952fig1highres.jpg" src="/files/ftp_upload/50952/50952fig1.jpg" /> 
Figure 1. General schematic of the steps involved in the generation of LN/fat pads chimeras and the analysis of the LNs recovered after transplant. To generate a chimeric eYFP- LNs-eYFP+ fat pads, the LN from an E18.5 eYFP+ fat pad is removed and replaced by a WT eYFP- newborn LN. The chimeric LN-fat pad is placed in culture for two days before transplantation under the kidney capsule of an adult WT host mouse. After 3 weeks, the chimera is recovered and the LN isolated for analysis.
<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50952/50952fig1highres.jpg" target="_blank">Click here to view larger image.</a>
<img alt="Figure 2" fo:content-width="4.75in" fo:src="/files/ftp_upload/50952/50952fig2highres.jpg" src="/files/ftp_upload/50952/50952fig2.jpg" /> 
Figure 2. Typical experimental results obtained using the LN/fat pad chimera method. A)&#160;Immunofluorescence staining of frozen sections of a LN recovered from a LN-EYFP+ fat pad chimera showing the colonization of the LN by host derived IgM+ B cells in red and CD4+ T cells in blue and the presence of EYFP+ cells in green (top panel). The fibroblastic stromal cell network is stained with Gp38/Podoplanin in blue and ERTR-7 in red. EYFP+ cells derived from the fat pads in green are integrated to the stromal cell network (middle, bottom panels and enlargement). B)&#160;Flow-cytometric analysis of single cell suspension of enzymatically digested LNs recovered from LN-EYFP+ fat pad chimeras stained with CD45, CD31 and Gp38/Podoplanin. Percentage of CD45- stromal cells is shown in the histogram on the top row. Percentages of CD31-Gp38+ fibroblastic stromal cells and CD31+Gp38- blood endothelial cells in the CD45- fraction are shown in the dot plot on the top row. Percentages of fat pad derived-EYFP+ cells in the CD31-Gp38+ fibroblastic stromal cell fraction (bottom row, left histogram) and in the CD31+Gp38- blood endothelial cell fraction (bottom row, right histogram) are shown. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50952/50952fig2large.jpg" target="_blank"> Click here to view larger figure</a>.
<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50952/50952fig2highres.jpg" target="_blank">Click here to view larger image.</a>

Watch this Scientific Journal Video about Generation of Lymph Node-fat Pad Chimeras for the Study of Lymph Node Stromal Cell Origin at JoVE.com

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

Generation of lymph node/fat pad chimeras for the study of lymph node stromal cell origin is described. The method involves the isolation of lymph nodes from newborn mice and embryonic fat pads, the generation of chimeric lymph node-fat pads, and their transfer under the kidney capsule of a host mouse.

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 ...

generation-lymph-node-fat-pad-chimeras-for-study-lymph-node-stromal

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

6.0K 조회수.  University of Birmingham. Generation of lymph node/fat pad chimeras for the study of lymph node stromal cell origin is described. The method involves the isolation of lymph nodes from newborn mice and embryonic fat pads, the generation of chimeric lymph node-fat pads, and their transfer under the kidney capsule of a host mouse.

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

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 response and therefore, the development of effector cells. As such, LNs are key to fighting invading pathogens and maintaining health. The choice of LN to study is dictated by accessibility and the desired model; the inguinal lymph node is well situated and easily supports studies of biologically relevant models of skin and genital mucosal infection.
The inguinal LN, like all LNs, has an extensive microvascular network supplying it with blood. In general, this microvascular network includes the main feed arteriole of the LN that subsequently branches and feeds high endothelial venules (HEVs). HEVs are specialized for facilitating the trafficking of immune cells into the LN during both homeostasis and infection. How HEVs regulate trafficking into the LN under both of these circumstances is an area of intense exploration. The LN feed arteriole, has direct upstream influence on the HEVs and is the main supply of nutrients and cell rich blood into the LN. Furthermore, changes in the feed arteriole are implicated in facilitating induction of adaptive immune response. The LN microvasculature has obvious importance in maintaining an optimal blood supply to the LN and regulating immune cell influx into the LN, which are crucial elements in proper LN function and subsequently immune response. 
The ability to study the LN microvasculature in vivo is key to elucidating how the immune system and the microvasculature interact and influence one another within the LN. Here, we present a method for in vivo imaging of the inguinal lymph node. We focus on imaging of the microvasculature of the LN, paying particular attention to methods that ensure the study of healthy vessels, the ability to maintain imaging of viable vessels over a number of hours, and quantification of vessel magnitude. Methods for perfusion of the microvasculature with vasoactive drugs as well as the potential to trace and quantify cellular traffic are also presented. 
Intravital microscopy of the inguinal LN allows direct evaluation of microvascular functionality and real-time interface of the direct interface between immune cells, the LN, and the microcirculation. This technique potential to be combined with many immunological techniques and fluorescent cell labelling as well as manipulated to study vasculature of other LNs.

A technique for performing intravital microscopy of the inguinal lymph node (LN) is outlined. Such technique allows for real-time, in vivo study of the lymph node microvasculature and structure both during homeostasis and infection. This technique can be adapted to cell trafficking studies and to other lymph node sites.

사타구니 림프 노드의 Intravital 현미경

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 ...

연구

면역학 및 감염병학

Non-surgical Intratracheal Instillation of Mice with Analysis of Lungs and Lung Draining Lymph Nodes by Flow Cytometry

Phagocytic cells such as alveolar macrophages and lung dendritic cells (LDCs) continuously sample antigens from the alveolar spaces in the 
lungs. LDCs, in particular, are known to migrate to the lung draining lymph nodes (LDLNs) where they present inhaled antigens to T cells initiating an 
appropriate immune response to a variety of immunogens1,2. To model interactions between the lungs and airborne antigens in mice, antigens can be 
administered intranasally1,3,4, intratracheally5 or as aerosols6. Delivery by each route involves distinct technical skills and limitations that need to be 
considered before designing an experiment. For example, intranasal and aerosolized exposure delivers antigens to both the lungs and the upper respiratory 
tract. Hence antigens can access the nasal associated lymphoid tissue (NALT)7, potentially complicating interpretation of the results. In addition, swallowing, 
sneezing and the breathing rate of the mouse may also lead to inconsistencies in the doses delivered. Although the involvement of the upper respiratory tract may 
be preferred for some studies, it can complicate experiments focusing on events specifically initiated in the lungs. In this setting, the intratracheal (i.t) 
route is preferable as it delivers test materials directly into the lungs and bypasses the NALT. Many i.t injection protocols involve either blind intubation of the 
trachea through the oral cavity or surgical exposure of the trachea to access the lungs. Herein, we describe a simple, consistent, non-surgical method for i.t 
instillation. The opening of the trachea is visualized using a laryngoscope and a bent gavage needle is then inserted directly into the trachea to deliver the 
innoculum. We also describe procedures for harvesting and processing of LDLNs and lungs for analysis of antigen trafficking by flow cytometry.

We illustrate non-surgical delivery of test materials into the lungs of anesthetized mice via the trachea. This method permits lung exposure to bacterial and viral pathogens, cytokines, antibodies, beads, chemicals, or dyes. We further describe harvesting and processing of lungs and lung draining lymph nodes (LDLNs) for flow cytometry.

유동세포계측법에 의해 폐, 폐 배수 림프절의 분석과 마우스의 비 수술 Intratracheal 떨어뜨림

Phagocytic cells such as alveolar macrophages and lung dendritic cells (LDCs) continuously sample antigens from the alveolar spaces in the 
lungs. LDCs, ...

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 of T cells into lymph nodes is a complex process which involves both cellular and chemical factors including chemokines. Recently, the use of two-photon microscopy has permitted to track T cells in intact lymph nodes and to derive some quantitative information on their behavior and their interactions with other cells. While there are obvious advantages to an in vivo system, this approach requires a complex and expensive instrumentation and provides limited access to the tissue. To analyze the behavior of T cells within murine lymph nodes, we have developed a slice assay 
1, originally set up by neurobiologists and transposed recently to murine thymus 2. In this technique, fluorescently labeled T cells are plated on top of an acutely prepared lymph node slice. In this video-article, the localization and migration of T cells into the tissue are analyzed in real-time with a widefield and a confocal microscope. The technique which complements in vivo two-photon microscopy offers an effective approach to image T cells in their natural environment and to elucidate mechanisms underlying T cell migration.

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

This protocol describes a method to image fluorescent T cells introduced into lymph node slices. The technique permits real-time analyses of T cell migration with traditional widefield fluorescence or confocal microscopes.

위드 필드와 공촛점 현미경과 Murine 림프절의 조각에서 T 세포의 생체내 이미징 전

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

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.

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.

Murine 외면적인 림프절 외과

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 ...

Intravital Imaging of the Mouse Popliteal Lymph Node

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign antigens from peripheral tissues to prime the adaptive immune response. Juxtaposed between innate and adaptive immune responses, the LN is an ideal site to study immune cell interactions1,2. Lymphocytes (T cells, B cells and NK cells), dendritic cells (DCs), and macrophages comprise the bulk of bone marrow-derived cellular elements of the LN. These cells are strategically positioned in the LN to allow efficient surveillance of self antigens and potential foreign antigens3-5. The process by which lymphocytes successfully encounter cognate antigens is a subject of intense investigation in recent years, and involves an integration of molecular contacts including antigen receptors, adhesion molecules, chemokines, and stromal structures such as the fibro-reticular network2,6-12. 
Prior to the development of high-resolution real-time fluorescent in vivo imaging, investigators relied on static imaging, which only offers answers regarding morphology, position, and architecture. While these questions are fundamental in our understanding of immune cell behavior, the limitations intrinsic with this technique does not permit analysis to decipher lymphocyte trafficking and environmental clues that affect dynamic cell behavior. Recently, the development of intravital two-photon laser scanning microscopy (2P-LSM) has allowed investigators to view the dynamic movements and interactions of individual cells within live LNs in situ12-16. In particular, we and others have applied this technique to image cellular behavior and interactions within the popliteal LN, where its compact, dense nature offers the advantage of multiplex data acquisition over a large tissue area with diverse tissue sub-structures11,17-18. It is important to note that this technique offers added benefits over explanted tissue imaging techniques, which require disruption of blood, lymph flow, and ultimately the cellular dynamics of the system. Additionally, explanted tissues have a very limited window of time in which the tissue remains viable for imaging after explant. With proper hydration and monitoring of the animal's environmental conditions, the imaging time can be significantly extended with this intravital technique. Here, we present a detailed method of preparing mouse popliteal LN for the purpose of performing intravital imaging.

Recent advances in 2-photon microscopy have enabled real-time in situ imaging of live tissues in animal models, thereby enhancing our ability to investigate cellular behavior in both physiologic and pathologic conditions. Here, we outline the preparations required to perform intravital imaging of the mouse popliteal lymph node.

마우스 오금 림프절의 Intravital 이미징

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

Isolation of Murine Lymph Node Stromal Cells

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host. 
Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.

Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells.

쥐과 림프절 기질 세포의 분리

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

The Mesenteric Lymph Duct Cannulated Rat Model: Application to the Assessment of Intestinal Lymphatic Drug Transport

The intestinal lymphatic system plays key roles in fluid transport, lipid absorption and immune function. Lymph flows directly from the small intestine via a series of lymphatic vessels and nodes that converge at the superior mesenteric lymph duct. Cannulation of the mesenteric lymph duct thus enables the collection of mesenteric lymph flowing from the intestine. Mesenteric lymph consists of a cellular fraction of immune cells (99% lymphocytes), aqueous fraction (fluid, peptides and proteins such as cytokines and gut hormones) and lipoprotein fraction (lipids, lipophilic molecules and apo-proteins). The mesenteric lymph duct cannulation model can therefore be used to measure the concentration and rate of transport of a range of factors from the intestine via the lymphatic system. Changes to these factors in response to different challenges (e.g., diets, antigens, drugs) and in disease (e.g., inflammatory bowel disease, HIV, diabetes) can also be determined. An area of expanding interest is the role of lymphatic transport in the absorption of orally administered lipophilic drugs and prodrugs that associate with intestinal lipid absorption pathways. Here we describe, in detail, a mesenteric lymph duct cannulated rat model which enables evaluation of the rate and extent of lipid and drug transport via the lymphatic system for several hours following intestinal delivery. The method is easily adaptable to the measurement of other parameters in lymph. We provide detailed descriptions of the difficulties that may be encountered when establishing this complex surgical method, as well as representative data from failed and successful experiments to provide instruction on how to confirm experimental success and interpret the data obtained.

Here we describe a technique to cannulate the mesenteric lymph duct in rats which enables quantification of lipid and drug transport via the lymphatic system following intestinal delivery. The technique can be adapted to assess mesenteric lymph concentrations and/or transport of fluid, immune cells, peptides, proteins and lipophilic molecules.

장간막 림프 덕트 유관 쥐 모델 : 창자 림프 약물 운송의 평가에 응용

The intestinal lymphatic system plays key roles in fluid transport, lipid absorption and immune function. Lymph flows directly from the small intestine ...

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

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly motile parasite not only reaches the liver to invade hepatocytes and transform into erythrocyte-infective form. It also migrates into the skin and to the proximal lymph node draining the injection site, where it can be recognized and degraded by resident and/or recruited myeloid cells. Intravital imaging reported the early recruitment of brightly fluorescent Lys-GFP positive leukocytes in the skin and the interactions between sporozoites and CD11c+ cells in the draining lymph node. We present here an efficient procedure to recover, identify and enumerate the myeloid cell subsets that are recruited to the mouse skin and draining lymph node following intradermal injection of immunizing doses of sporozoites in a murine model. Phenotypic characterization using multi-parametric flow cytometry provides a reliable assay to assess early dynamic cellular changes during inflammatory response to Plasmodium infection.

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

We describe here a protocol for isolating myeloid cells from mouse skin and draining lymph node following intradermal injection of Plasmodium sporozoites. Flow cytometry of collected cells provides a reliable assay to characterize the skin and draining lymph node inflammatory response to the parasite.

약독 화와 피내 접종에 따라 마우스의 피부와 배수 림프절에서 골수 세포 분리<em&gt; 변형체</em&gt; 포자 소체

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

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph node (DLN). The mobilization of DCs to the DLN is complex and remains to be fully elucidated during infection. Herein described is the use of an innovative, simple assay that relies on the fluorochrome 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) to track the migration of DCs during footpad infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin (BCG) in C57BL/6 mice. This assay enables the characterization of skin DC sub-populations that actively relocate to the draining, popliteal LN in response to BCG. This protocol originates from a BCG model where migratory skin DCs were identified by flow cytometry. The assay is amiable to the study and identification of DCs or other cells that home to the popliteal LN after inoculation of microbes, their metabolites or other inflammatory stimuli in the footpad, and consequently to study factors that regulate the migration of these cells.

An assay in mice to track cell migration from skin to draining lymph node is described which enables the characterization of skin Dendritic cells mobilized to the lymph node after footpad infection with Bacille Calmette-Gu&#233;rin.

와 감염시 배수 림프절에 쥐 피부 수지상 세포의 마이그레이션을 연구하는 CFSE 기반 분석<em&gt; 소 결핵균</em&gt; Bacille 칼 메트 - GUERIN

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

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

Large animals (both livestock and wildlife) serve as important reservoirs of zoonotic pathogens, including Brucella, Mycobacterium bovis, Salmonella, and E. coli, and are useful for the study of pathogenesis and/or spread of the bacteria in natural hosts. With the key function of lymph nodes in the host immune response, lymph node tissues serve as a potential source of RNA for downstream transcriptomic analyses, in order to assess the temporal changes in gene expression in cells over the course of an infection. This article presents an overview of the process of lymph node collection, tissue sampling, and downstream RNA processing in livestock, using cattle (Bos taurus) as a model, with additional examples provided from the American bison (Bison bison). The protocol includes information about the location, identification, and removal of lymph nodes from multiple key sites in the body. Additionally, a biopsy sampling methodology is presented that allows for a consistency of sampling across multiple animals. Several considerations for sample preservation are discussed, including the generation of RNA suitable for downstream methodologies like RNA-sequencing and RT-PCR. Due to the long delays inherent in large animal vs. mouse time course studies, representative results from bison and bovine lymph node tissues are presented to describe the time course of the degradation in this tissue type, in the context of a review of previous methodological work on RNA degradation in other tissues. Overall, this protocol will be useful to both veterinary researchers beginning transcriptome projects on large animal samples and to molecular biologists interested in learning techniques for in vivo tissue sampling and in vitro processing.

This protocol provides an overview of procedures for the isolation of RNA for the transcriptomic profiling of lymph node tissues from large animals, including steps in the identification and excision of lymph nodes from livestock and wildlife, sampling approaches to provide consistency across multiple animals, and considerations plus representative results for the post-collection preservation and processing for RNA analysis.

수집 및 RNA 분석에 대 한 큰 동물에서 림프절의 처리: 큰 동물 종의 림프 노드 Transcriptomic 연구에 대 한 준비

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