Name: determination of regulatory t cell subsets in murine thymus, pancreatic draining lymph node and spleen using flow cytometry
Uploaded: 2019-02-27T12:30:00
Description: Watch this Scientific Journal Video about Determination of Regulatory T Cell Subsets in Murine Thymus, Pancreatic Draining Lymph Node and Spleen Using Flow Cytometry at JoVE.com

The present study was supported financially by the Swedish Research Council, EXODIAB, the Swedish Diabetes Foundation, the Swedish Child Diabetes Fund, SEB Diabetesfonden and O.E. och Edla Johanssons vetenskapliga stiftelse. Authors would also like to thanks Per-Ola Carlsson and Stellan Sandler for their supports and discussions.

The local animal ethics committee at Uppsala University approved the animal experiments.
1. Harvesting Organs from Animals
<ol>
	<li>Sacrifice the mice by cervical dislocation.</li>
	<li>Place thymic glands and spleens in 20 mL scintillation vials or 15 mL conical tubes filled with 5 mL of Hanks&#180; balanced salt solution (HBSS). Place PDLNs in 1.5 mL micro tubes filled with 1 mL of RPMI 1640. Use the whole thymus and spleen, and all the PDLNs. 
	NOTE: Organs should b...

<ol>
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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=Expression+of+Helios,+an+Ikaros+transcription+factor+family+member,+differentiates+thymic-derived+from+peripherally+induced+Foxp3++T+regulatory+cells.">Expression of Helios, an Ikaros transcription factor family member, differentiates thymic-derived from peripherally induced Foxp3+ T regulatory cells.</a> The Journal of Immunology. 184 (7), 3433-3441 (2010).</li><li>Weiss, J. 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=Neuropilin+1+is+expressed+on+thymus-derived+natural+regulatory+T+cells,+but+not+mucosa-generated+induced+Foxp3++T+reg+cells.">Neuropilin 1 is expressed on thymus-derived natural regulatory T cells, but not mucosa-generated induced Foxp3+ T reg cells.</a> Journal of Experimental Medicine. 209 (10), 1723-1742 (2012).</li><li>Yadav, 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=Neuropilin-1+distinguishes+natural+and+inducible+regulatory+T+cells+among+regulatory+T+cell+subsets+in+vivo.">Neuropilin-1 distinguishes natural and inducible regulatory T cells among regulatory T cell subsets in vivo.</a> Journal of Experimental Medicine. 209 (10), S1711-S1719 (2012).</li><li>Singh, K., Hjort, M., Thorvaldson, L., Sandler, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concomitant+analysis+of+Helios+and+Neuropilin-1+as+a+marker+to+detect+thymic+derived+regulatory+T+cells+in+naive+mice.">Concomitant analysis of Helios and Neuropilin-1 as a marker to detect thymic derived regulatory T cells in naive mice.</a> Scientific Reports. 5, 7767 (2015).</li><li>Jungblut, M., Oeltze, K., Zehnter, I., Hasselmann, D., Bosio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+single-cell+suspensions+from+mouse+spleen+with+the+gentleMACS+Dissociator.">Preparation of single-cell suspensions from mouse spleen with the gentleMACS Dissociator.</a> Journal of Visualized Experiments. 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In this study, we isolated single cells from thymic glands, PDLNs and spleens of NOD mice, and investigated the expression of Helios and Nrp1 in CD4+CD8-CD25+Foxp3+ Treg cells using flow cytometry. In the present study, NOD mice were used, which is a murine model of type 1 diabetes. In a previous study, we have used the wild type mouse strains CD-1 and C57BL/6 mice to investigate whether Helios or Nrp1 is a better marker for distinguishing tTreg cells from pTreg cells. In that ...

Regulatory T (Treg) cells are critical for the homeostasis of immune system. Treg cells are defined by the expression of surface antigens CD4 and CD25, and the transcription factor forkhead box P3 (Foxp3)1,2,3. Sakaguchi et al. first showed that CD25 is constitutively expressed on T suppressor cells in mice4, which provided a pioneering observation for the identification of human Treg cells. Treg cells play a central role in immune tolerance by exerting a suppressive ability via a variety of mechanisms, including cytolysis through the secretion of granzymes to induce apoptosis, cytokine consumption and inhibition through the expression of cytotoxic T-lymphocyte antigen 45. Additionally, they can secrete inhibitory cytokines such as transforming growth factor beta (TGF-&#946;), interleukin-10 (IL-10) and IL-355. Treg cells can naturally be derived from the thymus, in which case they are designated thymic derived Treg (tTreg) cells, or they can be induced in the peripheral organs in that case are called peripherally induced Treg (pTreg) cells.
Helios, a member of the Ikaros transcription factor family, was reported to be a marker for distinguishing between tTreg cells and pTreg cells6. Later, two other groups reported that Neuropilin 1 (Nrp1), a semaphorin III receptor, could serve as a surface marker for tTreg cells under certain conditions7,8. Nevertheless, Singh et al. have shown that Helios is a better marker in this context in na&#239;ve mice9.
The subsets of Treg cells play a pivotal role in the maintenance of the homeostasis of the immune system and in the protection against autoimmunity and infection. Therefore, it is important to determine the numbers and proportions of these populations in both lymphoid and non-lymphoid tissues. To achieve this, one has to prepare single cell suspensions from these organs. A number of protocols have been described or used in previous studies. Mainly, automatic dissociators have been used in previously published reports10,11. Using automatic dissociators is convenient and time saving, but it is an expensive procedure. Therefore, we have used a manual method to prepare single cell suspensions from murine thymic glands, pancreatic draining lymph nodes (PDLNs) and spleens from normoglycemic non-obese diabetic (NOD) mice, and isolated single cells from these organs. This method has been used in our previous studies and we found that the manual method for preparing single cell suspension is as efficient as automatic dissociator method9,12,13,14. Furthermore, we used flow cytometry to determine the proportions of CD4+CD8-CD25+Foxp3+ Treg cells and the subsets of Treg cells tTreg and pTreg cells. The tTreg and pTreg cells were distinguished using Helios and Nrp1 as markers for tTreg cell. Thus, our results indicate that the manual method for preparing the single cells does work efficiently and the prepared single cell suspensions can be further used for studies using flow cytometry.

<table>
	<tbody>
		<tr>
			<td>NOD mice</td>
			<td></td>
			<td></td>
			<td>In-house breading</td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Statens veterin&#228;rmedicinska anstalt</td>
			<td>991750</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI-1640</td>
			<td>Sigma-Aldrich</td>
			<td>R0883-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>NH4Cl</td>
			<td>EMD Millipore</td>
			<td>1011450500</td>
			<td></td>
		</tr>
		<tr>
			<td>eBioscience Foxp3 / Transcription Factor Staining Buffer Set</td>
			<td>ThermoFisher</td>
			<td>00-5523-00</td>
			<td>Contains Fixation/Permeabilization Concentrate , Fixation/Permeabilization Diluent and Permeabilization Buffer (10X)</td>
		</tr>
		<tr>
			<td>eBioscience Flow Cytometry Staining Buffer</td>
			<td>ThermoFisher</td>
			<td>00-4222-26</td>
			<td></td>
		</tr>
		<tr>
			<td>CD4 Monoclonal Antibody (RM4-5), FITC, eBioscience</td>
			<td>ThermoFisher</td>
			<td>11-0042-85</td>
			<td></td>
		</tr>
		<tr>
			<td>CD25 Monoclonal Antibody (PC61.5), PE, eBioscience</td>
			<td>ThermoFisher</td>
			<td>12-0251-83</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Pharmingen&#160;APC-H7 Rat anti-Mouse CD8a</td>
			<td>BD Biosciences</td>
			<td>560182</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Neuropilin-1 APC-conjugated Antibody</td>
			<td>R&#38;D Systems</td>
			<td>FAB5994A</td>
			<td></td>
		</tr>
		<tr>
			<td>FOXP3 Monoclonal Antibody (FJK-16s), PE-Cyanine7, eBioscience</td>
			<td>ThermoFisher</td>
			<td>25-5773-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Pacific Blue anti-mouse/human Helios Antibody</td>
			<td>BioLegend</td>
			<td>137220</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 5 mL Round Bottom Polystyrene Test Tube, with Cell Strainer Snap Cap</td>
			<td>CORNING</td>
			<td>352235</td>
			<td>A 35 &#181;m nylon mesh is incorporated into the dual-position snap cap</td>
		</tr>
		<tr>
			<td>Tube 15ml, 120x17mm, PP</td>
			<td>Sarstedt</td>
			<td>62.554.002</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro tube 1.5ml</td>
			<td>Sarstedt</td>
			<td>72.690.001</td>
			<td></td>
		</tr>
		<tr>
			<td>disposable scintillation vials</td>
			<td>WHEATON</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometry</td>
			<td>BD</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometry analysis software</td>
			<td>Inivai Technologies</td>
			<td>Flowlogic</td>
			<td></td>
		</tr>
	</tbody>
</table>

determination of regulatory t cell subsets in murine thymus, pancreatic draining lymph node and spleen using flow cytometry

Our immune system consists of a number and variety of immune cells including regulatory T cells (Treg) cells. Treg cells can be divided into two subsets, thymic derived Treg (tTreg) cells and peripherally induced Treg (pTreg) cells. They are present in different organs of our body and can be distinguished by specific markers, such as Helios and Neuropilin 1. It has been reported that tTreg cells are functionally more suppressive than pTreg cells. Therefore, it is important to determine the proportion of both tTreg and pTreg cells when investigating heterogeneous cell populations. Herein, we collected thymic glands, pancreatic draining lymph nodes and spleens from normoglycemic non-obese diabetic mice to distinguish tTreg cells from pTreg cells using flow cytometry. We manually prepared single cell suspensions from these organs. Fluorochrome conjugated surface CD4, CD8, CD25, and Neuropilin 1 antibodies were used to stain the cells. They were kept in the fridge overnight. On the next day, the cells were stained with fluorochrome conjugated intracellular Foxp3 and Helios antibodies. These markers were used to characterize the two subsets of Treg cells. This protocol demonstrates a simple but practical way to prepare single cells from murine thymus, pancreatic draining lymph node and spleen and use them for subsequent flow cytometric analysis.

To investigate the expression of Nrp1 and Helios on tTreg and pTreg cells, we prepared single cells from thymic glands, PDLNs and spleens of normoglycemic NOD mice and stained them with the Treg cell markers CD4, CD25, Foxp3, Helios and Nrp1 for flow cytometric analysis. The results were analyzed as shown in the representative gating strategies (Figure 1). We found that the proportion of Helios+ cells among CD4+CD8-CD25+<...

Watch this Scientific Journal Video about Determination of Regulatory T Cell Subsets in Murine Thymus, Pancreatic Draining Lymph Node and Spleen Using Flow Cytometry at JoVE.com

Determination of Regulatory T Cell Subsets in Murine Thymus, Pancreatic Draining Lymph Node and Spleen Using Flow Cytometry

Herein, we present a protocol to prepare single cells from murine thymus, pancreatic draining lymph node and spleen to further study these cells using flow cytometry. In addition, this protocol was used for determining the subsets of regulatory T cells using flow cytometry.

Our immune system consists of a number and variety of immune cells including regulatory T cells (Treg) cells. Treg cells can be divided into two subsets, ...

This protocol helps in investigating the role of immune cells in particular organs in health and pathology. The main advantage of this technique is that this is a cost-effective technique since it's a manual technique. Demonstrating the procedure will be Zhengkang Luo, a PhD student from my laboratory.To begin, place the thymic glands and spleens from previously euthanized mice into 20 ml scintillation phials, or 15 ml conical tubes, filled with 5 ml of Hank's balanced salt solution. Place the pancreatic draining lymph nodes into 1.5 ml micro-tubes filled with 1 ml of RPMI-1640. Make sure to use the whole thymus and spleen, and all of the PDLNs.Keep the organs on ice throughout the entire procedure. Using a pair of scissors, thoroughly squeeze the thymus and spleen to release the immune cells. Discard the remaining thymic and splenic capsules.Transfer the cell suspension to a 15 ml conical tube. Centrifuge at 433 times g and at 4 degrees celsius for five minutes, and discard the supernatant. To lyse the red blood cells, re-suspend the cell suspension in 5 ml of 0.2 Molar ammonium chloride, and incubate at room temperature for ten minutes.Invert the tubes gently every two minutes. At the end of the incubation, add 5 ml of HBSS to stop the lysis. Centrifuge the tubes at 433 times g and at 4 degrees celsius for five minutes.Discard the supernatant, and re-suspend the cells in approximately 5 ml of HBSS. Then, fill the tubes with HBSS. Repeat this process, from centrifuging the samples to filling the tube with HBSS, one time.Centrifuge the tubes once more at 433 times g and at four degrees celsius for five minutes. Discard the supernatant, and re-suspend the pellet in HBSS. Obtain 5 ml round-bottom tubes with cell-strainer caps.Transfer 1 ml of the thymic cell suspension and 500 microlitres of the splenic cell suspension to the tubes by applying the suspension to the cell-strainer caps. First, place a 15 ml conical tube into a rack. Place a sterile 250 micrometer metal mesh over the tube.Rinse the mesh with 1 ml of RPMI. Next, transfer the lymph nodes to the metal mesh and use a pair of tweezers to grind them through the mesh. Apply 1 ml of RPMI on the mesh to flush the cells into the tube.Repeat this process of transferring and grinding the lymph nodes three times for each sample, and then remove the mesh. Centrifuge the tubes at 433 times g and at four degrees celsius for five minutes. Discard the supernatant and re-suspend the cells in approximately 5 ml of RPMI.Then, fill the tubes with RPMI. Repeat this process, from centrifuging the samples to filling the tube with RPMI, one time. Centrifuge the tubes again at 433 times g and at four degrees celsius for five minutes.Discard the supernatant and re-suspend the cell pellet in 2 ml of RPMI. Transfer 2 ml of the cell suspension into 5 ml round-bottom tubes with cell-strainer caps. First, centrifuge the cell suspension from the thymus, spleen and PDLN samples at 433 times g and at four degrees celsius for five minutes.Discard the supernatant. Stain the cells with surface antibodies as outlined in the text protocol, and incubate the tubes on ice for 40 minutes. Next, add 200 microliters of FACS buffer to each tube.Centrifuge at 433 times g and at four degrees celsius for five minutes, and discard the supernatant. Repeat this process, adding the buffer, centrifuging, and discarding the supernatant, one time. Re-suspend the cell pellet in 500 microliters of permeabilization fixation buffer to fix and permeabilize the cells.Transfer the tubes to a refrigerator at four degrees celsius overnight. The next day, centrifuge the tubes at 433 times g and at four degrees celsius for five minutes. Discard the supernatant, and re-suspend the cell pellet in 500 microliters of permeabilization washing buffer.Centrifuge the cells again at 433 times g and at four degrees celsius for five minutes and discard the supernatant. Stain the cells with intracellular antibodies, as outlined in the text protocol. Incubate the tubes on ice for 1 hour.After this, add 500 microliters of permeabilization washing buffer to each tube. Centrifuge at 433 times g and at four degrees celsius for five minutes. Discard the supernatant, and re-suspend the pellet in 500 microliters of permeabilization washing buffer.Centrifuge once more at 433 times g and at four degrees celsius for five minutes. Discard the supernatant and re-suspend the cell pellet in 300 microliters of FACS buffer. Then, analyze the cells on a flow cytometer, as outlined in the text protocol.In this study, single cells are isolated from thymic glands, PDLNs and spleens of normal glycemic NOD mice, and are stained with Treg cell markers, CD4, CD25, Foxp3, Helios, and neuropilin-1 for flow cytometric analysis. The results are analyzed and are shown here as representative gating strategies. The proportion of Helios-positive cells among the CD4-positive, CD8-negative, CD25-positive, Foxp3-positive Treg cells is seen to be higher than that of Nrp1-positive cells in all three organs.More than 80%of Treg cells in the thymus are seen to express Helios, which is higher than in the PDLN, and the spleen. The proportion of NrP1-positive cells among Helios-positive Treg cells is seen to be higher in the PDLN than those in either the thymus or spleen. The majority of Nrp1-positive Treg cells also express Helios, and the proportion of Helios-positive cells among Nrp1 positive Treg cells is seen to be higher in the thymus and spleen than in the PDLN.Together, these results indicate that Helios is a better marker to detect T Treg cells than Nrp1. Lymph nodes are small, but some intracellular markers require a large number of cells to give a good signal in flow cytometry. Other methods cannot be performed after this procedure because the cells are already stained and dead.But the animal's four cell markers can be replaced to study other types of immune cells.

determination-regulatory-t-cell-subsets-murine-thymus-pancreatic

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Immunology and Infection

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 ...

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

It is now well known that T lymphocytes play a critical role in the development of several cardiovascular diseases1,2,3,4,5. For example, studies from our group have shown that hypertension is associated with an excessive accumulation of T cells in the vessels and kidney during the development of experimental hypertension6. Once in these tissues, T cells produce several cytokines that affect both vascular and renal function leading to vasoconstriction and sodium and water retention1,2. To fully understand how T cells cause cardiovascular and renal diseases, it is important to be able to identify and quantify the specific T cell subsets present in these tissues. T cell subsets are defined by a combination of surface markers, the cytokines they secrete, and the transcription factors they express. The complexity of the T cell population makes flow cytometry and intracellular staining an invaluable technique to dissect the phenotypes of the lymphocytes present in tissues. Here, we provide a detailed protocol to identify the surface and intracellular markers (cytokines and transcription factors) in T cells isolated from murine kidney, aorta and aortic draining lymph nodes in a model of angiotensin II induced hypertension. The following steps are described in detail: isolation of the tissues, generation of the single cell suspensions, ex vivo stimulation, fixation, permeabilization and staining. In addition, several fundamental principles of flow cytometric analyses including choosing the proper controls and appropriate gating strategies are discussed.

This article provides detailed methodology to identify and quantify functional T lymphocyte subsets present within murine kidney, aorta and lymph nodes by intracellular staining and flow cytometry. The model of angiotensin II induced hypertension was chosen to explain, step-by-step, the procedures and fundamental principles of flow cytometry and intracellular staining.

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

Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal of immunosuppression. The mixed leukocyte reaction (MLR), limiting dilution assays, and trans-vivo delayed-type hypersensitivity (DTH) assay have all been applied to this question, but these methods have limited predictive ability and/or significant practical limitations that reduce their usefulness. Imaging flow cytometry is a technique that combines the multiparametric quantitative powers of flow cytometry with the imaging capabilities of fluorescent microscopy. We recently made use of an imaging flow cytometry approach to define the proportion of recipient T cells capable of forming mature immune synapses with donor antigen-presenting cells (APCs). Using a well-characterized mouse heart transplant model, we have shown that the frequency of in vitro immune synapses among T-APC membrane contact events strongly predicted allograft outcome in rejection, tolerance, and a situation where transplant survival depends on induced regulatory T cells. The frequency of T-APC contacts increased with T cells from mice during acute rejection and decreased with T cells from mice rendered unresponsive to alloantigen. The addition of regulatory T cells to the in vitro system reduced prolonged T-APC contacts. Critically, this effect was also seen with human polyclonally expanded, naturally occurring regulatory T cells, which are known to control the rejection of human tissues in humanized mouse models. Further development of this approach may allow for a deeper characterization of the alloreactive T-cell compartment in transplant recipients. In the future, further development and evaluation of this method using human cells may form the basis for assays used to select patients for immunosuppression minimization, and it can be used to measure the impact of tolerogenic therapies in the clinic.

This paper describes a method for measuring alloreactivity in a mixed population of T cells using imaging flow cytometry.

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Characterization of Thymus-dependent and Thymus-independent Immunoglobulin Isotype Responses in Mice Using Enzyme-linked Immunosorbent Assay

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a formidable defense against invading pathogens via diverse mechanisms. One major goal of vaccination is to induce protective antigen-specific antibodies to prevent life-threatening infections. Both thymus-dependent (TD) and thymus-independent (TI) antigens can elicit robust antigen-specific IgM responses and can also induce the production of isotype-switched antibodies (IgG, IgA and IgE) as well as the generation of memory B cells with the help provided by antigen presenting cells (APCs). Here, we describe a protocol to characterize TD and TI Ig isotype responses in mice using enzyme-linked immunosorbent assay (ELISA). In this protocol, TD and TI Ig responses are elicited in mice by intraperitoneal (i.p.) immunization with hapten-conjugated model antigens TNP-KLH (in alum) and TNP-polysaccharide (in PBS), respectively. To induce TD memory response, a booster immunization of TNP-KLH in alum is given at 3 weeks after the first immunization with the same antigen/adjuvant. Mouse sera are harvested at different time points before and after immunization. Total serum Ig levels and TNP-specific antibodies are subsequently quantified using Ig isotype-specific Sandwich and indirect ELISA, respectively. In order to correctly quantify the serum concentration of each Ig isotype, the samples need to be appropriately diluted to fit within the linear range of the standard curves. Using this protocol, we have consistently obtained reliable results with high specificity and sensitivity. When used in combination with other complementary methods such as flow cytometry, in vitro culture of splenic B cells and immunohistochemical staining (IHC), this protocol will allow researchers to gain a comprehensive understanding of antibody responses in a given experimental setting.

In this paper, we describe a protocol to characterize T-dependent and T-independent immunoglobulin (Ig) isotype responses in mice using ELISA. This method used alone or in combination with flow cytometry will allow researchers to identify differences in B cell-mediated Ig isotype responses in mice following T-dependent and T-independent antigen immunization.

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a ...