Name: myeloid cell isolation from mouse skin and draining lymph node following intradermal immunization with live attenuated plasmodium sporozoites
Uploaded: 2016-05-18T13:00:00
Description: Watch this Scientific Journal Video about Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites at JoVE.com

The authors thank Patricia Baldacci, Vanessa Lagal and Sabine Thiberge for critical reading, Irina Dobrescu and Sabine Thiberge for help in taking pictures and Pauline Formaglio for teaching in vivo imaging of sporozoite motility in the mouse skin. We also would like to thank Marek Szatanik and the Center for Production and Infection of Anopheles (CEPIA-Institut Pasteur) for mosquito rearing. This study was supported by the AXA Research Fund&#160;and funds from the Laboratoire d&#8217;Excellence &#8220;Integrative Biology of Emerging Infectious Diseases&#8221; (grant no. ANR-10-LABX-62-IBEID).

All procedures were approved by the committee of the Pasteur Institute and by the local Ethics Committee on Animal Experimentation (Ethical committee IDF-Paris 1, Paris, France; agreement number: 2012-0015) and performed in accordance with the applicable guidelines and regulations.&#160;
1. Materials and Reagents
<ol>
	<li>Use female Anopheles stephensi mosquitoes (Sda500 strain) that feed on infected mice 3-5 days after emergence and rear as described previously21.</li>
	<li>Use parasites <e...

<ol>
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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=Sporozoite+immunization+of+human+volunteers+under+chemoprophylaxis+induces+functional+antibodies+against+pre-erythrocytic+stages+of+Plasmodium+falciparum.">Sporozoite immunization of human volunteers under chemoprophylaxis induces functional antibodies against pre-erythrocytic stages of Plasmodium falciparum.</a> Malar J. 13, 136 (2014).</li><li>Amino, R., 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=Quantitative+imaging+of+Plasmodium+transmission+from+mosquito+to+mammal.">Quantitative imaging of Plasmodium transmission from mosquito to mammal.</a> Nat Med. 12 (2), 220-224 (2006).</li><li>Yamauchi, L. 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B., Zavala, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD8++T+lymphocytes+protective+against+malaria+liver+stages+are+primed+in+skin-draining+lymph+nodes.">CD8+ T lymphocytes protective against malaria liver stages are primed in skin-draining lymph nodes.</a> Nat Med. 13 (9), (2007).</li><li>Obeid, M., 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=Skin-draining+lymph+node+priming+is+sufficient+to+induce+sterile+immunity+against+pre-erythrocytic+malaria.">Skin-draining lymph node priming is sufficient to induce sterile immunity against pre-erythrocytic malaria.</a> EMBO Mol Med. 5 (2), 250-263 (2013).</li><li>Amino, R., 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=Host+cell+traversal+is+important+for+progression+of+the+malaria+parasite+through+the+dermis+to+the+liver.">Host cell traversal is important for progression of the malaria parasite through the dermis to the liver.</a> Cell Host Microbe. 3 (2), 88-96 (2008).</li><li>Radtke, A. 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B., 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=Early+skin+immunological+disturbance+after+Plasmodium-infected+mosquito+bites.">Early skin immunological disturbance after Plasmodium-infected mosquito bites.</a> Cell Immunol. 277 (1-2), 22-32 (2012).</li><li>Mac-Daniel, L., 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=Local+immune+response+to+injection+of+Plasmodium+sporozoites+into+the+skin.">Local immune response to injection of Plasmodium sporozoites into the skin.</a> J Immunol. 193 (3), 1246-1257 (2014).</li><li>Ribeiro-Gomes, F. L., Peters, N. C., Debrabant, A., Sacks, D. 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In the perspective of large-scale vaccination of humans using a whole sporozoite malaria vaccine, one of the main challenges to overcome is to develop optimized routes and methods of parasite administration to ensure successful immunization and protection24,25. In humans, the evaluation of protective efficacy mediated by live attenuated parasites (LAP) has been performed following natural mosquito bites2, as well as intradermal, subcutaneous25,26 and IV immunizations27. As repo...

Malaria is one of the deadliest infectious diseases in the world, killing more than half a million people per year. Infection by Plasmodium, the causal agent of the disease, begins by a pre-erythrocytic (PE) phase. During this phase, sporozoites injected into the host skin by a female Anopheline mosquito reach the liver via the bloodstream and differentiate inside hepatocytes into the parasite forms that infect red blood cells and cause the symptoms of the disease.
The PE stages of Plasmodium represent a privileged target for anti-malaria vaccination. Indeed, live attenuated vaccines against these stages, such as radiation attenuated sporozoites (RAS), genetically arrested parasites (GAP) or chemoprophylaxis and sporozoites (CPS) have demonstrated their capacity to protect both rodent and human hosts1-9. In the rodent model, most vaccination studies are conducted utilizing intravenous immunization, which is the gold standard in terms of protective efficacy. However, the description of a skin stage and the importance of the skin-associated draining lymph node (dLN) in eliciting protection has changed our perception of the PE phase and emphasized the importance of the intradermal route of injection. Intravital imaging of P. berghei sporozoites injected into the skin of rodents has shown that only ~25% of the inoculum reaches the liver via the bloodstream. The remaining ~75% distributes between the proximal dLN (~15%) and the skin (~50%)10,11, where a small proportion can transform and remain alive for weeks inside skin cells12,13. Moreover, subsequent studies described that the establishment of effective protective immunity after intradermal immunization mainly takes place in the skin-dLN, where parasite specific CD8+ T cells are activated, and only marginally in the spleen or liver-dLNs14,15.
While most studies have concentrated on the characterization of the effector cells implicated in the establishment of protective immune response, much less is known about the fate of live attenuated parasites injected into the skin, especially their interactions with the innate immune system. In particular, characterization of antigen-presenting cells involved in parasite antigen uptake, processing and presentation to CD8+ T cells is of critical importance, knowing that PE antigen acquisition can occur both in the skin and dLN compartments. Previous intravital imaging studies described an early influx of brightly fluorescent Lys-GFP positive cells in the skin following an infectious mosquito bite16 while early interactions between sporozoites and dendritic cells were observed in the dLN10,17. More recently, it has been reported that sporozoites inoculated in the skin by mosquitoes increases the motility of both dendritic and regulatory T cells in the skin of mice, while a decrease number of antigen presenting cells was observed in the dLN18.
We aimed to identify and quantify more precisely the leukocyte subsets recruited in the skin and corresponding dLN as well as those interacting with the parasite following intradermal injection of immunizing doses of RAS19. In this context, we isolated myeloid cells (CD45+CD11b+) from both tissues and characterized subpopulations of interest by multi=parametric flow cytometry. Consistent with the immune response described in the early stage of Leishmania major skin infection20, the primary host response to sporozoite injection consists of a successive recruitment of polymorphonuclear neutrophils (CD45+CD11b+Ly6G+Ly6Cint) followed by inflammatory monocytes (CD45+CD11b+Ly6G-Ly6C+) which are identified on the basis of differential expression of the Ly6G and Ly6C surface markers.
We describe here a protocol for isolating myeloid cells from the mouse skin and dLN following intradermal injection of immunizing doses of RAS extracted from infected mosquito salivary glands. Reproducible intradermal injections and tissue processing are critical steps to quantify phenotypic changes of infiltrating cell population within infected tissues. The approach detailed below provides a reliable assay to assess the skin and dLN inflammatory response to Plasmodium parasite and can be extended to various experimental systems.

<table>
	<tbody>
		<tr>
			<td>Ketamine: Imalgene&#174; 1000</td>
			<td>Merial</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Xylazine: Rompun&#174; 2%</td>
			<td>Bayer</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NanoFil syringe + 35 gauge needle</td>
			<td>World Precision Instruments&#160;</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Omnican&#174; 50 Insulin syringe 0,5 ml/50 I.U.</td>
			<td>B. Braun Medical&#160;</td>
			<td>9151125</td>
			<td>-</td>
		</tr>
		<tr>
			<td>MultiwellTM 6 well tissue culture plate &#8211; Flat Bottom</td>
			<td>BD Falcon&#160;</td>
			<td>353046</td>
			<td>-</td>
		</tr>
		<tr>
			<td>70 &#181;m cell strainer&#160;</td>
			<td>BD Falcon&#160;</td>
			<td>352350</td>
			<td>-</td>
		</tr>
		<tr>
			<td>2 ml syringe</td>
			<td>Terumo</td>
			<td>SS-02S</td>
			<td>-</td>
		</tr>
		<tr>
			<td>BLUE MAXTM 15ml Polypropylene conical tube</td>
			<td>BD Falcon&#160;</td>
			<td>352097</td>
			<td>-</td>
		</tr>
		<tr>
			<td>BLUE MAXTM 50ml Polypropylene conical tube</td>
			<td>BD Falcon&#160;</td>
			<td>352098</td>
			<td>-</td>
		</tr>
		<tr>
			<td>5ml Polystyrene Round-Bottom Tube with 35&#181;m Cell-Strainer Cap</td>
			<td>BD Falcon&#160;</td>
			<td>352235</td>
			<td>-</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS 1X Cacl2- and MgCl2-free</td>
			<td>Life Technologies</td>
			<td>14190-094</td>
			<td>-</td>
		</tr>
		<tr>
			<td>DMEM 1X + GlutaMAXTM</td>
			<td>Life Technologies</td>
			<td>31966-021</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Collagenase from Clostridium histolyticum, Type IV 0.5-5.0 FALGPA units/mg solid&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>C5138</td>
			<td>400 U/ml&#160;</td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I from bovine pancreas, type IV&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>D5025</td>
			<td>50 &#181;g/ml</td>
		</tr>
		<tr>
			<td>EDTA disodium salt</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>E-5134</td>
			<td>10mM or 2.5 mM</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Biowest</td>
			<td>S1810-500</td>
			<td>-</td>
		</tr>
		<tr>
			<td>HEPES buffer solution (1M)</td>
			<td>Gibco</td>
			<td>15630-056</td>
			<td>25 mM</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue Stain (0,4%)</td>
			<td>Life Technologies</td>
			<td>15250-061</td>
			<td>Dilution 1:10&#160;</td>
		</tr>
		<tr>
			<td>Anti-mouse CD16/CD32 (2.4G2 clone)</td>
			<td>BD Biosciences</td>
			<td>553142</td>
			<td>10&#181;g/ml final (1:50)</td>
		</tr>
		<tr>
			<td>DAPI FluoroPureTM grade</td>
			<td>Life Technologies</td>
			<td>D21490</td>
			<td>1&#181;g/ml final</td>
		</tr>
		<tr>
			<td>Anti-mouse CD45 (30-F11 clone)</td>
			<td>BD Biosciences</td>
			<td>559864</td>
			<td>&#160;Dilution 1:200</td>
		</tr>
		<tr>
			<td>Anti-mouse CD11b (M1/70 clone)</td>
			<td>BD Biosciences</td>
			<td>557657</td>
			<td>&#160;Dilution 1:400</td>
		</tr>
		<tr>
			<td>Anti-mouse CD8&#945; (5H10 clone)</td>
			<td>Life Technologies</td>
			<td>MCD0830</td>
			<td>&#160;Dilution 1:100</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Female C57BL/6JRj mice (7-week-old)&#160;</td>
			<td>Janvier Laboratories</td>
			<td>-</td>
			<td>-</td>
		</tr>
	</tbody>
</table>

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.

We recently demonstrated that needle-syringe injection of immunizing doses of P. berghei sporozoites in the mouse skin induces a successive recruitment of polymorphonuclear neutrophils followed by inflammatory monocytes in the skin and dLN19. The Protocol Section described above details the procedure used to successfully isolate live myeloid cells from both tissues following multiple injections of large number of sporozoites in the ear dermis (Figures 1 </stron...

Watch this Scientific Journal Video about Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites at JoVE.com

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.

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 ...

The overall goal of this set of procedures is to immunize mice with radiation attenuated plasmodium parasites. To facilitate the analysis of myeloid cell recruitment in the mouse skin and draining lymph nodes. This method can help answer key questions in the malaria field, such as what are the cellular actors involved in the innate immunoresponse to live attenuated parasites.Between 18-25 days after the infections blood meal, collect and cold anesthetize infected mosquitoes in a 15 milliliter tube on ice. Next, carefully invert the tube into a Petri dish on ice and expose the insects to a 12 kilorad dose of gamma or X-irradiation. Collect the irradiated mosquito salivary glands in 15 microliters of DPBS on ice.And then, gently crush the glands to release the sporozoites. The injection of a highly concentrated parasite suspension in a small volume is critical, so it is important to harvest a sufficient number of salivary glands from well-infected mosquitoes as evidenced by robust expression of green fluorescence. Re-suspend the parasite suspension prior to filtration through a 35 micrometer strainer adapted to a 1.5 milliliter microcentrifuge tube.Using a microscope counting chamber, count the total number of isolated sporozoites and adjust the parasite suspension to an 8.3 times ten to the fourth sporozoites per microliter concentration in cold DPBS on ice. Before injecting the sporozoites, confirm the proper level of sedation of the experimental animal by toe pinch and apply eye ointment to prevent corneal drying. Then, working quickly, use clear tape to gently fix the ventral side of the ear pinna under a stereo microscope and load a ten microliter syringe, equipped with a 35 gauge needle, with 0.6 microliters of resuspended parasites.Next, carefully insert the needle, bevel side up, beneath the epidermis on the dorsal side of the ear and inject 0.15 microliters of sporozoites into the site. A characteristic papule will be observed at the injection site. After injecting the dermis three more times, as just demonstrated, carefully remove the tape and monitor the mouse until it is fully recovered.At the desired time point following sporozoite inoculation, sacrifice the mouse and start the procedure immediately. Make sure the mouse demonstrates signs of clinical death. Disinfect the ear and the corresponding neck region with 70 percent ethanol.Next, using sterile scissors and forceps, make an incision in the jugulo-carotidian area. Then stretch the incision with two forceps to expose the grey-looking draining lymph node. Carefully pinch and remove the connective tissue directly above the lymph node and extract it with a second pair of forceps, placed underneath it.Transfer the lymph node into a 70 micron strainer in one well of a six-well tissue culture plate, containing 4.5 milliliters of standard medium, supplemented with collagenase and DNase. Next, harvest the entire inoculated ear and use two forceps to pinch and separate the ear into the dorsal and ventral aspects. Then place each side of the ear into a well of a second six-well plate, containing 4.5 milliliters per well of medium supplemented with digestive enzymes, taking care that each tissue is completely immersed in the medium.To isolate the immune cells from the lymph nodes, incubate the plate of lymphoid tissue at 37 degrees Celsius and five percent CO2 under gentle agitation. After 15 minutes, add ten millimolar EDTA to each well and place the plate on ice. Next, for each lymph node, use the end of a new five millimeter syringe plunger and press the lymph node against the strainer using circular motions to dissociate the tissue.When only white connective tissue is left, rinse the strainer two times with 500 microliters of DPBS containing FBS and EDTA and transfer the wash to a 15 milliliter conical tube. Then, wash the well two more times with 500 microliters of the supplemented DPBS and transfer the wash to the tube placed on ice. To isolate the immune cells from the ear, incubate the ear leaflets at 37 degrees Celsius and five percent CO2 with gentle agitation.After 20 minutes, cut the tissues into smaller pieces to facilitate their digestion and return the samples to the incubator. After 40 minutes, stop the digestion with ten millimolar EDTA and place the plate on ice. Using a one milliliter pipette with a cut tip, transfer the entire contents of each well into new wells containing 70 micron strainers.And use a five milliliter syringe plunger to dissociate the ear tissue fragments with circular motions as just demonstrated. When only white connective tissue and hair remain, wash the strainers and wells with DPBS plus FBS and EDTA as just demonstrated, pooling the washes into a 50 milliliter tube on ice. Finally, wash the well twice with supplemented DPBS and transfer the wash to the tube placed on ice.In this video, the migration of radiation attenuated sporozoites into the mouse skin 15 minutes after intradermal injection of the parasites can be seen. 24 hours after intradermal sporozoite injection, total cells are isolated from the skin and draining lymph node. As observed on this figure, the frequency of myeloid cells in the skin and draining lymph node is robustly increased compared to naive mice.Once mastered, the first steps of the protocol for mosquito irradiation to sporozoite injection can be completed in four hours. While attempting this procedure, it is important to remember that sporozoite isolated from mosquito salivary glands rapidly lose their infectivity. So they must be injected as quickly as possible.After watching this video, you should have a good understanding of how to isolate myeloid cells from the mouse skin and draining lymph node to explore the immunoresponse to intradermal injection of live attenuated plasmodium parasites.

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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 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.

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 ...

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.

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.

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.

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.

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 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.

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 ...

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.

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

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune cells in rodent models of skin inflammation. Here, we describe a protocol for the digestion of mouse skin in a nutrient-rich solution with collagenase D, followed by separation of hematopoietic cells using a discontinuous density gradient. Cells thus obtained can be used for in vitro studies, in vivo transfer, and other downstream cellular and molecular analyses including flow cytometry. This protocol is an effective and economical alternative to expensive mechanical dissociators, specialized separation columns, and harsher trypsin- and dispase-based digestion methods, which may compromise cellular viability or density of surface proteins relevant for phenotypic characterization or cellular function. As shown here in our representative data, this protocol produced highly viable cells, contained specific immune cell subsets, and had no effect on integrity of common surface marker proteins used in flow cytometric analysis.

This protocol describes enzymatic digestion of mouse skin in nutrient-rich medium followed by gradient separation to isolate leukocytes. Cells thus derived can be used for diverse downstream applications. This is an effective, economical, and improved alternative to tissue dissociation machines and harsher trypsin and dispase-based tissue digestion protocols.

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune ...

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.

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