The authors would like to thank Dr. Alain Trautmann who encouraged us to perform lymph node slices. This work was supported in part by grants from the Ligue Nationale Contre le Cancer, the Fondation pour la Recherche M&eacute;dicale en France and the Association pour la recherche sur le Cancer.

1. Preparing slices from lymph nodes
<ol>
 <li>Prepare a 4% low-melting point agarose solution for embedding. Dilute agarose in PBS and microwave this solution. When the agarose is completely dissolved, maintain the solution at 37&deg;C until ready for use.</li>
 <li>Prepare a 6-well tissue culture plate. Add 1.1 ml RPMI complete culture medium per well, and insert an organotypic filter in each well. Place the 6-well plate at 4&deg;C until ready to transfer slices.</li>
 <li>Place stainless steel washers, with inner diameters of 4 mm, into a plastic dish filled with RPMI complete medium. Washers will be further used to concentrate the cells on the vibratome cut slice.</li>
 <li>Sacrifice the mouse in accordance with local animal welfare regulations. Carefully remove the peripheral lymph nodes from the surrounding tissue and place them in a plastic dish containing ice-cold PBS. Care must be taken not to damage the lymph node structure, as these organs are very soft.</li>
 <li>Pour the 4%-agarose gel into a 35-mm plastic dish and delicately transfer the nodes into the gel. Leave the agarose gel on ice for 5 min to harden. </li>
 <li>Remove the block from the dish and trim the agar in order to leave 3-5 mm of gel around each lymph node. </li>
 <li>Attach the embedded nodes on the specimen disk of the vibratome with non-toxic tissue glue. Install the specimen disk in the tray filled with ice-cold PBS.</li>
 <li>Section the agar-embedded tissue at 320 &mu;m thickness with the vibratome speed set on a slow range (0.3 mm/s) and the vibration frequency set on a medium range (1.5 mm).</li>
 <li>Carefully transfer lymph node slices as they are being cut onto the organotypic culture inserts using fine forceps. Place 3 slices on each insert. Use great care as slices can be easily damaged. A typical peripheral lymph node will yield 5 sections when cut at 320 &mu;m. Discard the first and last slices, as they only contain superficial tissue.</li>
 <li>Place stainless steel washers on each individual slice. Make sure washers are well positioned on the agarose surrounding the tissue. </li>
 <li>Incubate the culture plate at 37&deg;C in a 5% CO2 humidified incubator until ready to plate T cells.</li>
</ol>
2. Isolating T cells from mouse lymph nodes
<ol>
<li>Isolate T cells according to the previously published JoVE article 3.</li>
<li>Use T cells immediately and proceed to the next section. Otherwise, lymphocytes might be cultured overnight in complete RPMI-medium supplemented with 10 ng/ml IL-7. </li>
</ol>
3. Labeling of isolated T cells
<ol>
<li>Wash cells in HBSS. Resuspend them in the same buffer at 10 x 106 per ml.</li>
<li>Add the same volume of HBSS containing 1 &mu;M Cell Tracker green CMFDA giving a final concentration of 0.5 &mu;M.</li>
<li>Incubate the cell suspension at 37&deg;C during 5 min. Wash cells twice with complete RPMI-medium. </li>
<li>Resuspend cells at a final concentration of 10 x 106 per ml in complete RPMI-medium. These labeled cells will be plated onto the slices.</li>
</ol>
Other fluorescent dyes than CMFDA might be used too, but keep in mind that these molecules are potentially toxic above certain concentrations. 
4. Plating labeled T cells onto lymph node slices
<ol>
<li>Remove excess medium contained in the inner part of the washer with a pipette. Do not allow the tissue to become dry; work quickly during this step.</li>
<li>Gently dispense 10 to 20 &mu;l labeled T cells that corresponds to 1 to 2 x 105 cells in the inner part of the washer. Care must be taken not to touch the tissue with the tip of the pipette. Be sure that the drop of cell suspension stays in place. </li>
<li>Place the slices with cells in incubator at 37&deg;C and 5% CO2 for at least 30 min to allow lymphocytes to migrate into the tissue. </li>
</ol>
5. Imaging T cells within lymph node slices
In this video-article, we image fluorescent T cells in lymph node slices with a widefield inverted microscope and a confocal upright microscope. 
<ol>
 <li>Set the temperature at 37&deg;C. Our microscopes are equipped with temperature-controlled chambers. </li>
 <li>Install a perfusion system enabling continuous perfusion of the lymph node slice with oxygenated (5% CO2, 95 % O2) phenol red-free RPMI medium. In our system, perfused media enters into the imaging chamber from one side by gravity. The medium is aspirated with a pump connected to a waste collection flask. Set the media flow rate to 1 ml/min.</li>
 <li>With fine forceps, delicately take out the slice from the 6-well plate and dip the preparation in warmed oxygenated RPMI medium for a few seconds to get rid of fluorescent cells not infiltrated into the tissue.</li>
</ol> 
Imaging T cells with a wide-field inverted microscope
<ol start="4"> 
<li>Place the slice upside down on a custom-made imaging chamber which consists of nylon threads glued onto the inner part of a glass bottom dish. In this chamber, the nylon threads elevate the slice from the glass and enable the oxygenated solution to diffuse into the tissue. This is required as T cell migration in lymph nodes is strongly dependent on oxygen. Secure the slice by adding onto it a stainless steel washer. In these conditions, the slice lies a few microns above the bottom of the dish, which facilitates renewal of the perfusion solution.</li>
<li>To capture a large field of view (800 x 800 &mu;m, approximately one third of the node's slice surface), collect images using a 10x dry objective lens. We found that the 10x Nikon S fluor 0.5 NA was most suitable for our analyses. </li>
</ol>
A typical time-lapse imaging experiment captures 5 optical planes spanning a total depth of 50 &mu;m in the axial (z) dimension. Fluorescent T cells are usually imaged at intervals ranging 10 to 30 seconds during 10 to 20 min. 
Imaging T cells with a confocal upright microscope
The confocal microscope used in this protocol is a Leica SP5 equipped with a 20x water immersion objective (Olympus, 20x/0.95 NA). 
<ol start="6">
<li>Secure the slice to the imaging stage of the microscope. Note: We usually place a washer on the preparation to immobilize it.</li>
<li>Set an imaging session by collecting a series of images along the z axis. To image T cells in an intact tissue structure, the start position is usually set at 5 to 10 &mu;m below the first labeled T cell. </li>
</ol>
6. Representative results
By following this video-protocol you should expect to visualize a large number of fluorescent T cells accumulated in the T zone of the node, a phenomenon that normally occurs in this particular environment in vivo (Figure 1). In particular, T cells should be excluded from B cell zones, which are usually just beneath the capsule. A successful experiment will also result in T cells recruited into the tissue (Figure 2), displaying a highly motile behavior (Figure 3) consistent with published results obtained in intact lymph nodes 4. On average, the mean velocity of individual T cells within slices should be close to 10 &mu;m/min (Figure 4). 
<img src="/files/ftp_upload/3054/3054fig1.jpg" alt="Figure 1"/> Figure 1. T cells accumulate in a lymph node slice. Fluorescently-labeled T cells (CMFDA, green) were added to a lymph node slice 30 min before image acquisition (top picture). The image is the maximum projection of 5 images spanning 50 &mu;m in the z direction. The bright field image is shown in bottom. Images were captured with a widefield microscope. 
<img src="/files/ftp_upload/3054/3054fig2.jpg" alt="Figure 2"/> Figure 2. T cells are recruited into a lymph node slice. Fluorescently-labeled T cells (CMFDA, green) were added to a lymph node slice 30 min before image acquisition using a confocal microscope. Images were captured at the cut surface (top picture) and 40 &mu;m below (bottom picture). 
<img src="/files/ftp_upload/3054/3054fig3.jpg" alt="Figure 3"/> Figure 3. T cells are highly motile within a lymph node slice. Fluorescently-labeled T cells were imaged during 12 min using a widefield microscope. Trajectories of individual T cells are displayed as color-coded tracks to represent increasing displacements from blue (low motile cells) to red (high motile cells). Tracks were calculated using Imaris software. The white line follows the edge of the node, whereas the dashed oval delimits the putative B cell zone. 
<img src="/files/ftp_upload/3054/3054fig4.jpg" alt="Figure 4"/> Figure 4. Within a lymph node slice, the T cell velocity is close to 10 &mu;m/min. The speeds were calculated using Imaris software from tracks represented in Figure 3.

<ol>
	<li>Asperti-Boursin, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CCR7+ligands+control+basal+T+cell+motility+within+lymph+node+slices+in+a+phosphoinositide+3-kinase-independent+manner.">CCR7 ligands control basal T cell motility within lymph node slices in a phosphoinositide 3-kinase-independent manner.</a> J Exp Med. 204, 1167-1167 (2007).</li><li>Bhakta, N. R., Oh, D. Y., Lewis, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+oscillations+regulate+thymocyte+motility+during+positive+selection+in+the+three-dimensional+thymic+environment.">Calcium oscillations regulate thymocyte motility during positive selection in the three-dimensional thymic environment.</a> Nat Immunol. 6, 143-143 (2005).</li><li>Matheu, M. P., Cahalan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+CD4++T+cells+from+mouse+lymph+nodes+using+Miltenyi+MACS+purification.">Isolation of CD4+ T cells from mouse lymph nodes using Miltenyi MACS purification.</a> J Vis Exp. , (2007).</li><li>Miller, M. J., Wei, S. H., Parker, I., Cahalan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+imaging+of+lymphocyte+motility+and+antigen+response+in+intact+lymph+node.">Two-photon imaging of lymphocyte motility and antigen response in intact lymph node.</a> Science. 296, 1869-1869 (2002).</li><li>Ehrlich, L. I., Oh, D. Y., Weissman, I. L., Lewis, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+contribution+of+chemotaxis+and+substrate+restriction+to+segregation+of+immature+and+mature+thymocytes.">Differential contribution of chemotaxis and substrate restriction to segregation of immature and mature thymocytes.</a> Immunity. 31, 986-986 (2009).</li><li>Gollmer, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CCL21+mediates+CD4++T-cell+costimulation+via+a+DOCK2/Rac-dependent+pathway.">CCL21 mediates CD4+ T-cell costimulation via a DOCK2/Rac-dependent pathway.</a> Blood. 114, 580-580 (2009).</li></ol>

No conflicts of interest declared.

We have described a straightforward, quick and robust technique for generating lymph node slices, which are used to investigate the behavior of introduced T cells. In recent years, this method has been applied successfully using thymic and lymph node slices to identify the extracellular factors controlling T cell positioning and motility 1,5. It has also been used to measure Ca2+ responses in thymocytes during positive selection and in T cells upon antigen recognition 2,6. The overlay slice assay presents advantages and limitations that deserve to be discussed. Of note, this system permits access to the tissue, useful if one needs to manipulate slices pharmacologically in order to interfere with the molecular control of cell migration. Subsequent to imaging, the slices can be processed for immunohistochemistry to collect further information about the structures that have been imaged. Moreover, observation can be made with a traditional widefield fluorescence microscope. Although the resolution is not as good as with a confocal or a two-photon microscope and that phototoxicity is in principle more severe with one-photon than with two-photon microscopy, it has the advantages of simplicity, a lower cost, and larger choice of excitation wavelengths. 
The imaging of T cells in an intact lymph node requires intravenous injection of labeled lymphocytes that subsequently home to lymphoid organs. As we have shown previously 1, the slice assay is perfectly compatible with adoptive transfer experiment. However, the length of time needed for T cells to home into lymph nodes can complicate the use of fluorescent dyes that have a tendency to leak out of the cells over time. This is the case of the Ca2+ dye fura-2. With the fast recruitment (&lt; 30 min) of T cells into the tissue, the slice assay provides an opportunity to use such dyes. Finally, this method has the great advantage to analyze T cell functions in several human tissues kept alive. 
This experimental system presents also limitations that need to be kept in mind. Damage associated with the slicing may affect T cell functioning, especially in the superficial region of the tissue near the cut surface. In order to assess cell death within the slice, we have used the fluorescent dye SYTOX green that penetrates cells with compromised plasma membrane. Our experiments reveal that about 20% of total nodal cells, mostly localized in the superficial region of the tissue, were fluorescently labeled with this nuclear dye. Another potential concern with the assay is the ability of the slices to retain important soluble factors including chemokines. Although, we have no indication that our data were affected by such problems since T cells display a good motility within the slice, we would like to stress the importance of imaging T cells in healthy regions located at several tens of microns from the cut surface. Whereas, this could be done with traditional microscopes (widefield or confocal) like in this protocol, it is likely that the lymph node slice preparation combined with two-photon imaging will increase the spatial resolution in depth and reduce the phototoxicity.

<table border="1">
	<tbody>
		<tr>
			<th>Name of the reagent</th>
			<th>Company</th>
			<th>Catalogue number</th>
			<th>Comments (optional)</th>
		</tr>
		<tr>
			<td>Confocal microscope </td>
			<td>Leica</td>
			<td>SP5</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Vibratome </td>
			<td>Leica</td>
			<td>VT1200S</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fine Forceps </td>
			<td>World Precision Instruments</td>
			<td>14142</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>30 mm Culture inserts </td>
			<td>Millipore</td>
			<td>PICM0RG50</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>RPMI </td>
			<td>Invitrogen</td>
			<td>61870010</td>
			<td>Complete RPMI-medium is made by adding 10 % heat-inactivated fetal calf serum and Penicillin/streptomycin</td>
		</tr>
		<tr>
			<td>Hanks' Balanced Salt Solution (HBSS) </td>
			<td>Invitrogen</td>
			<td>14170088</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Low gelling temperature Agarose, type VII-A </td>
			<td>Sigma-Aldrich</td>
			<td>A0701</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Butyl Cyanoacrylate Glue, Vetbond</td>
			<td>3M</td>
			<td>1469</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Stainless steel washers, 4 mm of inner diameter </td>
			<td>DIY store</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cell Tracker green CMFDA </td>
			<td>Invitrogen</td>
			<td>C7025</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

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.

Watch this Scientific Journal Video about Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes at JoVE.com

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.

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 ...

ex-vivo-imaging-t-cells-murine-lymph-node-slices-with-widefield

Research

JoVE Journal

Immunology and Infection

16.8K 조회수.  Université Paris Descartes, CNRS (UMR 8104). 이 프로토콜은 림프절의 조각에 소개 이미지 형광 T 세포 수있는 방법을 설명합니다. 이 기술은 전통적인 위드 형광 또는 공촛점 현미경과 T 세포의 마이 그 레이션의 실시간 분석을 수 있습니다.

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

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

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 ...

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 ...

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter lesions in inflammatory demyelinating disorders like multiple sclerosis (MS). In these disorders, mainly CD8+ T-cells of putative specificity for myelin- and oligodendrocyte-related antigens are found, so that neuronal apoptosis in grey matter lesions may be a collateral effect of these cells. Different types of animal models are established to study the underlying mechanisms of the mentioned pathophysiological processes. However, although they mimic some aspects of MS, it is impossible to dissect the exact mechanism and time course of &#8216;&#8216;collateral&#8217;&#8217; neuronal cell death. To address this course, here we show a protocol to study the mechanisms and time response of neuronal damage following an oligodendrocyte-directed CD8+ T cell attack. To target only the myelin sheath and the oligodendrocytes, in vitro activated oligodendrocyte-specific CD8+ T-cells are transferred into acutely isolated brain slices. After a defined incubation period, myelin and neuronal damage can be analysed in different regions of interest. Potential applications and limitations of this model will be discussed.

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

To address mechanisms of demyelination and neuronal apoptosis in cortical lesions of inflammatory demyelinating disorders, different animal models are used. We here describe an ex vivo approach by using oligodendrocyte-specific CD8+ T-cells on brain slices, resulting in oligodendroglial and neuronal death. Potential applications and limitations of the model are discussed.

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter ...

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

To enter its human host, herpes simplex virus type 1 (HSV-1) must overcome the barrier of mucosal surfaces, skin, or cornea. HSV-1 targets keratinocytes during initial entry and establishes a primary infection in the epithelium, which is followed by latent infection of neurons. After reactivation, viruses can become evident at mucocutaneous sites that appear as skin vesicles or mucosal ulcers. How HSV-1 invades skin or mucosa and reaches its receptors is poorly understood. To investigate the invasion route of HSV-1 into epidermal tissue at the cellular level, we established an ex vivo infection model of murine epidermis, which represents the site of primary and recurrent infection in skin. The assay includes the preparation of murine skin. The epidermis is separated from the dermis by dispase II treatment. After floating the epidermal sheets on virus-containing medium, the tissue is fixed and infection can be visualized at various times postinfection by staining infected cells with an antibody against the HSV-1 immediate early protein ICP0. ICP0-expressing cells can be observed in the basal keratinocyte layer already at 1.5 hr postinfection. With longer infection times, infected cells are detected in suprabasal layers, indicating that infection is not restricted to the basal keratinocytes, but the virus spreads to other layers in the tissue. Using epidermal sheets of various mouse models, the infection protocol allows determining the involvement of cellular components that contribute to HSV-1 invasion into tissue. In addition, the assay is suitable to test inhibitors in tissue that interfere with the initial entry steps, cell-to-cell spread and virus production. Here, we describe the ex vivo infection protocol in detail and present our results using nectin-1- or HVEM-deficient mice.

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

The skin is one target tissue of the human pathogen herpes simplex virus type 1 (HSV-1). To explore the invasion route of HSV-1 into tissue, we established an ex vivo infection model of murine epidermal sheets which represent the outermost layer of skin.

To enter its human host, herpes simplex virus type 1 (HSV-1) must overcome the barrier of mucosal surfaces, skin, or cornea. HSV-1 targets keratinocytes ...

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been considered as the only source of &#945;&#946;T cells. Yet, thymic involution begins early in life leading to a drastically reduced output of na&#239;ve &#945;&#946;T cells into the periphery. Nevertheless, even centenarians can build immunity against newly acquired pathogens. Recent research suggests extrathymic &#945;&#946;T cell development, however our understanding of pathways that may compensate for thymic loss of function are still rudimental. &#947;&#948; T cells are innate lymphocytes that constitute the main T-cell subset in the tissues. We recently ascribed a so far unappreciated outstanding function to a &#947;&#948; T cell subset by showing that the scarce entity of CD4+ V&#948;1+&#947;&#948; T cells can transdifferentiate into &#945;&#946;T cells in inflammatory conditions. Here, we provide the protocol for the isolation of this progenitor from peripheral blood and its subsequent cultivation. V&#948;1 cells are positively enriched from PBMCs of healthy human donors using magnetic beads, followed by a second step wherein we target the scarce fraction of CD4+ cells with a further magnetic labeling technique. The magnetic force of the second labeling exceeds the one of the first magnetic label, and thus allows the efficient, quantitative and specific positive isolation of the population of interest. We then introduce the technique and culture condition required for cloning and efficiently expanding the cells and for identification of the generated clones by FACS analysis. Thus, we provide a detailed protocol for the purification, culture and ex vivo expansion of CD4+ V&#948;1+&#947;&#948; T cells. This knowledge is prerequisite for studies that relate to this &#945;&#946;T cell progenitor`s biology and for those who aim to identify the molecular triggers that are involved in its transdifferentiation.

Isolation and Ex Vivo Culture of V&amp;#948;1+CD4+&amp;#947;&amp;#948; T Cells, an Extrathymic &amp;#945;&amp;#946;T-cell Progenitor

Here, we provide an optimized protocol for the isolation and cloning of the scarce T-cell entity of peripheral V&#948;1+CD4+ T cells that is, as we showed recently, an extrathymic &#945;&#946; T-cell progenitor. This technique allows to quantitatively isolate, clone and efficiently expand these cells in ex vivo culture.

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been ...

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte cell culture and 64Cu-antibody receptor targeting of the cells. In vitro evaluation of the radiolabel and non-invasive in vivo cell tracking in an animal model of an airway delayed-type hypersensitivity reaction (DTHR) by PET/CT are described.
In detail, the conjugation of a mAb with the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) is shown. Following the production of radioactive 64Cu, radiolabeling of the DOTA-conjugated mAb is described. Next, the expansion of chicken ovalbumin (cOVA)-specific CD4+ interferon (IFN)-&#947;-producing T helper cells (cOVA-TH1) and the subsequent radiolabeling of the cOVA-TH1 cells are depicted. Various in vitro techniques are presented to evaluate the effects of 64Cu-radiolabeling on the cells, such as the determination of cell viability by trypan blue exclusion, the staining for apoptosis with Annexin V for flow cytometry, and the assessment of functionality by IFN-&#947; enzyme-linked immunosorbent assay (ELISA). Furthermore, the determination of the radioactive uptake into the cells and the labeling stability are described in detail. This protocol further describes how to perform cell tracking studies in an animal model for an airway DTHR and, therefore, the induction of cOVA-induced acute airway DHTR in BALB/c mice is included. Finally, a robust PET/CT workflow including image acquisition, reconstruction, and analysis is presented.
The 64Cu-antibody receptor targeting approach with subsequent receptor internalization provides high specificity and stability, reduced cellular toxicity, and low efflux rates compared to common PET-tracers for cell labeling, e.g.64Cu-pyruvaldehyde bis(N4-methylthiosemicarbazone) (64Cu-PTSM). Finally, our approach enables non-invasive in vivo cell tracking by PET/CT with an optimal signal-to-background ratio for 48 h. This experimental approach can be transferred to different animal models and cell types with membrane-bound receptors that are internalized.

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo  Cell Trafficking by PET/CT

Following the preparation of a 64Cu-modified monoclonal antibody binding to a transgenic murine T cell receptor, T cells are radiolabeled in vivo, analyzed for viability, functionality, labeling stability and apoptosis, and adoptively transferred into mice with an airway delayed-type hypersensitivity reaction for non-invasive imaging by positron emission tomography/computed tomography (PET/CT).

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte ...