This research was supported by grants from National Defense Medical College and by a Health and Labour Sciences research grant for research on intractable diseases from the Ministry of Health, Labour and Welfare, Japan.

The experimental protocol was approved by the Animal Research Committee of National Defense Medical College (no. 16058). The animals were maintained on standard laboratory chow (CLEA Japan Inc, Tokyo, Japan). The laboratory animals were treated in accordance with National Institutes of Health guidelines.
1. Collection and separation of lymphocytes
NOTE: Since lymphocytes must be fresh and cannot be stored, they must be collected from rats for each experiment. In addition, gut-tropic lymphocytes must be collected directly from the thoracic duct where they circulate.&#160;All procedures are expected to be completed within 6 hours, and all instruments, gloves, and surfaces are clean. In order to keep the animals in a physiologically stable state, all saline and other fluids used in the experiment should be kept warm.
<ol>
	<li>Form a cannulation tube (1 mm diameter) like a heparin curve (a radius of about 5 mm) after dipping it in hot water (about 80&#8211;90 &#176;C) and fix it to a plastic board using adhesive tape for a few hours in advance.</li>
	<li>Anesthetize a male Wistar rat (8&#8211;12 weeks) with intraperitoneal injection of a mixture of Midazolam (2mg/kg), Domitor (0.15mg/kg), and Betulphar (2.5mg/kg) and cut the body hair of the abdomen using a hair clipper. Remove the hair firmly after shaving so that the hair does not enter the abdominal cavity.</li>
	<li>After confirming that the stable depth of anesthesia required for the procedure is obtained by toe pinch, make an incision with surgical scissors horizontally from midline to the left subcostal area. Be careful not to damage the blood vessels in the parietal peritoneum by watching the vessels under light.</li>
	<li>Wrap the stomach with wet gauze and move the stomach gently to the right outside of the body to reveal the retroperitoneal organs. Insert a notch between the erector muscles of the spine and the adipose tissue using surgical scissors and bluntly separate them with fingers.</li>
	<li>Carefully strip off the connective tissue around the thoracic duct. The excess connective tissue increases with age. Expose the thoracic duct from just beneath the left crus of the diaphragm caudally until a 20 mm length of the thoracic duct was visible. Gently separate adhesions between the thoracic duct and aorta using precision tweezers or wet cotton swabs.
	<ol>
		<li>Perform this process carefully because some rats have arterioles branching from the abdominal artery crossing over the thoracic duct or a short thoracic duct due to a higher position of the lymph plexus (small thoracic ducts spreading like spider webs). Avoid unnecessary contact to the thoracic duct to avoid inducing edema.</li>
	</ol>
	</li>
	<li>Apply a ligature by a string (3-0 silk) on the thoracic duct just beneath the left crus of the diaphragm. The caudal thoracic duct becomes distended.</li>
	<li>Make a hole (5 mm diameter) in the abdominal wall by stabbing the cutting edge of surgical scissors, pass the hairpin-curved cannulation tube, and ligate the tube on the iliopsoas muscle at one point. Fill the cannulation tube with normal saline containing 10 U/mL heparin.</li>
	<li>After placing a string (3-0 silk) beneath the distended thoracic duct, stab the thoracic duct with the sharply cut edge of a cannulation tube, cannulate it toward the tail about 5 mm, and ligate the thoracic duct with cannulation tube for fixation.</li>
	<li>To supply saline to prevent dehydration, create a hole (3 mm diameter) on the anterior wall of the antrum of the stomach using precision tweezers and pass the silicon tube (2 mm in diameter) to the duodenum through the pyloric ring. After stitching up a wound and maintaining the animals in Bollman&#8217;s cages, start to infuse sugar-laden saline into each rat&#8217;s duodenum from the silicon tube at a flow rate of 3 mL/h. Cover the entire cage with a paper towel to keep it warm.</li>
	<li>Fix the cannulation tube to the hole in the center of the lid of a 50-mL conical tube on ice containing 6 U/mL heparin, 10% fetal bovine serum, and RPMI 1640 medium (pH 7.4; see the table of materials), and collect thoracic duct lymphocytes (TDLs) on ice. Be careful not to contact the tip of the tube to the wall of the conical tube to prevent cannulated-tube occlusion due to fibrin clot formation inside the tube. Lymph fluid (about 20 mL) including about 1.0 x 108&#65374;109 lymphocytes can be obtained in 6 hours. To obtain the lymphocytes&#160; under complete anesthesia, plane of anesthesia is monitored and lymph flow is observed appropriately. The same anesthetics are added at the same dose about 3 hours after rats are put into Ballman&#39;s cage to obtain deep anesthesia continuously for 6 hours.</li>
</ol>
2. Lymphocyte labeling with carboxyfluorescein diacetate succinimidyl ester (CFDSE)
<ol>
	<li>Dissolve CFDSE (Table of Materials) in dimethyl sulfoxide (DMSO) to 15.6 mM (500 &#181;g of CFDSE dissolved in 60 &#181;L of DMSO).</li>
	<li>Incubate lymphocytes (1 x 108&#65374;109) in 50 mL of RPMI 1640 with 50 &#181;L of CFDSE solution for 30 min at 37 &#176;C as described previously10.</li>
</ol>
3. Experimental setup for microvascular studies
<ol>
	<li>Anesthetize recipient rats (8&#8211;12 weeks) with intraperitoneal injection of a mixture of Midazolam (2mg/kg), Domitor (0.15mg/kg), and Betulphar (2.5mg/kg) and confirm the depth of anesthesia by toe pinch. Wet the abdomen before grooming the fur to minimize hair contamination. Then open the abdomen of the recipient Wister rat via a midline incision.</li>
	<li>Put a rat on a portable stainless-steel plate (about 120 x 300 mm) with a rectangle hole around the center covered with a glass slide (24 x 50 mm). Choose about 10 cm of the ileal segment including PPs for observation.</li>
	<li>Keep the intestine as warm as possible and moist with phosphate buffered saline (PBS) warmed to 37 &#176;C. Soak gauze that is used to cover the small intestine with PBS.</li>
	<li>&#160;While giving continuous anesthesia with 2 % isoflurane (see table of materials),&#160;put the slide on the stage of the microscope and choose suitable areas for observation of the microcirculation in PPs where some HEVs are running through the serosa. PPs range in size; larger ones are suitable for observation of the microcirculation using CLSM. The small intestine is not straight in places; a straight segment at least 2 cm long without any tension is suitable for observation.</li>
	<li>Cover the adjacent intestinal segment and mesentery with absorbent cotton soaked with PBS. Place the intestine segment between two rolled cotton balls and position it as far as possible from the rat&#8217;s body to prevent it from being vibrated by the rat&#8217;s heartbeat and breathing.</li>
	<li>Using a 1 mL syringe, slowly inject (over 1 min) CFSE-labeled TDLs (1 x 108 cells) into the jugular vein of the recipient rats. A rapid intravenous injection may influence the systemic circulation.</li>
</ol>
4. Microcirculation of lymphocytes
<ol>
	<li>Continuously monitor TDLs in the microvasculature of PPs using CLSM and record on a computer for 3 hours using time-lapse photography at 30 s intervals. The depth from the serosa to the HEV of PPs is about 25 &#956;m, enabling the observation of the stroma and capillary lymph vessels to a depth of 30 &#956;m.</li>
	<li>Inject Texas Red&#8211;dextran (25 mg/kg) into the jugular vein of each recipient rat to stain the bloodstream and Hoechest 33342 (5 mg/kg) to stain the cell nuclei.</li>
	<li>(Optional) To quantify lymphocyte dynamics, define lymphocytes adhering to HEVs more than 30 seconds as &#8220;adhesive lymphocytes&#8221; and lymphocytes emigrating from HEVs to stroma as &#8216;&#8216;migrating lymphocytes&#8221;. Then calculate the average percentage of migration (migrating lymphocytes / adhesive lymphocytes + migrating lymphocytes) per field of vision (approximately 0.3 mm2).</li>
</ol>

<ol>
	<li>Miura, S., Hokari, R., Tsuzuki, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucosal+immunity+in+gut+and+lymphoid+cell+trafficking.">Mucosal immunity in gut and lymphoid cell trafficking.</a> Annals of Vascular Diseases. 5, 275-281 (2012).</li><li>Stein, J. V., Nombela-Arrieta, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemokine+control+of+lymphocyte+trafficking:+a+general+overview.">Chemokine control of lymphocyte trafficking: a general overview.</a> Immunology. 116, 1-12 (2005).</li><li>Yopp, A. 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=Sphingosine-1-phosphate+receptors+regulate+chemokine-driven+transendothelial+migration+of+lymph+node+but+not+splenic+T+cells.">Sphingosine-1-phosphate receptors regulate chemokine-driven transendothelial migration of lymph node but not splenic T cells.</a> The Journal of Immunology. 175, 2913-2924 (2005).</li><li>Lucke, S., Levkau, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+functions+of+sphingosine-1-phosphate.">Endothelial functions of sphingosine-1-phosphate.</a> Cellular Physiology and Biochemistry. 26, 87-96 (2010).</li><li>Bollman, J. L., Cain, J. C., Grindlay, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+for+the+collection+of+lymph+from+the+liver,+small+intestine,+or+thoracic+duct+of+the+rat.">Techniques for the collection of lymph from the liver, small intestine, or thoracic duct of the rat.</a> The Journal of Laboratory and Clinical. 333, 1349-1352 (1948).</li><li>Bhalla, D. K., Murakami, T., Owen, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microcirculation+of+Intestinal+Lymphoid+Follicles+in+Rat+Peyer's+Patches.">Microcirculation of Intestinal Lymphoid Follicles in Rat Peyer's Patches.</a> Gastroenterology. 81, 481-491 (1981).</li><li>Miura, S., 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=Intravital+Demonstration+of+Sequential+Migration+Process+of+Lymphocyte+Subpopulations+in+Rat+Peyer's+Patches.">Intravital Demonstration of Sequential Migration Process of Lymphocyte Subpopulations in Rat Peyer's Patches.</a> Gastroenterology. 109, 1113-1123 (1995).</li><li>Tomura, 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=Monitoring+cellular+movement+in+vivo+with+photoconvertible+fluorescence+protein+"Kaede"+transgenic+mice.">Monitoring cellular movement in vivo with photoconvertible fluorescence protein "Kaede" transgenic mice.</a> Proceeding of the National Academy of Sciences of the United States of America. 105, 10871-10876 (2008).</li><li>Schulz, O., 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=Hypertrophy+of+Infected+Peyer's+Patches+Arises+From+Global,+Interferon-Receptor,+and+CD69-independent+Shutdown+of+Lymphocyte+Egress.">Hypertrophy of Infected Peyer's Patches Arises From Global, Interferon-Receptor, and CD69-independent Shutdown of Lymphocyte Egress.</a> Mucosal Immunology. 7, 892-904 (2014).</li><li>Higashiyama, 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=P-Selectin-Dependent+Monocyte+Recruitment+Through+Platelet+Interaction+in+Intestinal+Microvessels+of+LPS-Treated+Mice.">P-Selectin-Dependent Monocyte Recruitment Through Platelet Interaction in Intestinal Microvessels of LPS-Treated Mice.</a> Microcirculation. 15, 441-450 (2008).</li><li>Shirakabe, K., 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=Amelioration+of+colitis+through+blocking+lymphopcytes+entry+to+Peyer's+patches+by+sphingosine-1-phosphate+lyase+inhibitor.">Amelioration of colitis through blocking lymphopcytes entry to Peyer's patches by sphingosine-1-phosphate lyase inhibitor.</a> Journal of Gastroenterology and Hepatology. 33, 1608-1616 (2018).</li><li>Cenk, S., Thorsten, R. M., Irina, B. M., Ulrich, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+Microscopy:+Visualizing+Immunity+in+Context.">Intravital Microscopy: Visualizing Immunity in Context.</a> Immunity. 21, 315-329 (2004).</li><li>Alex, Y. C. H., Hai, Q., Ronald, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Illuminating+the+Landscape+of+In+Vivo+Immunity:+Insights+from+Dynamic+In+Situ+Imaging+of+Secondary+Lymphoid+Tissues.">Illuminating the Landscape of In Vivo Immunity: Insights from Dynamic In Situ Imaging of Secondary Lymphoid Tissues.</a> Immunity. 21, 331-339 (2004).</li></ol>

The authors have nothing to disclose.

Here we described a protocol for collecting na&#239;ve gut-tropic lymphocytes and observing their migration in rat PPs. These procedures can reveal how lymphocytes move in the microvasculature of PPs and make it possible to visually compare their dynamics under a normal or medicated condition. The direct observation of these dynamics has much merit to obtain a clue for immunological modification by some drugs, although the observational period is limited to only a few hours.
We mentioned some tips in the methods. Among them, one of the most delicate procedures is that the operator must cannulate the thoracic duct quickly and precisely. If an operation takes a long time and involves unnecessary damage, significant bleeding and inflammation occur, resulting in a decreased TDL collection due to edema of the connective tissue and obstruction of the thoracic duct. We used to cannulate the tube after creating a small incision on the thoracic duct using scissors with a small cutting edge. However, this procedure interferes with the cannulation site due to draining lymph. Therefore, now we do not cut the thoracic duct; rather, we stab it with a sharp edge. This method makes the cannulation easier and increases the success rates. In addition, the incision site in the thoracic duct is very important because of the limited abdominal cavity. If the incision site is too close to the diaphragm, the curved part of the cannulation tube would bump against the diaphragm. If it is too far from the diaphragm, it is also difficult to cannulate the tube deeply enough to ensure fixation due to the lymph plexus of the caudal thoracic duct. Even in cases of good cannulation, the cannulation tube can be easily occluded by fibrin formation. Thus, we recommend periodically gently flushing the tube with heparin.
Recent advances in CLSM made it possible to observe the focused area more clearly and precisely in real time compared to a previous study11. We now use CLSM in our laboratory, but we can observe the microcirculation more clearly using a multiphoton microscope. Although the protocol requires modification in several points along with technical progress, it enables observation of the localized area of the organ of interest12,13.
The merit and key of the method is that it involves separating lymphocytes from recipient animals, incubating them using any kind of compounds in vitro, and observing them after their injection into recipient animals. This makes it possible to elucidate the effects of lymphocytes alone. On the contrary, if we want to elucidate the effects of any kind of compounds on cells other than lymphocytes, such as the vascular endothelium, we can treat recipient animals with any kind of compounds and observe them after the injection of control lymphocytes. To elucidate the same things using mice, we must create conditional genetically manipulated animals one by one. In addition, sorting the isolated lymphocytes by using specific surface marker would make it possible to study the dynamics of specific types of lymphocytes.
As shown in the video of this study, lymphocytes move indefinitely and laterally through the vascular endothelium of the actual body, so it is difficult to evaluate effects on their behavior using in vivo observations only. Furthermore, if the mechanism of action of an administered drug is to be examined, it is originally unsuitable for examining each site of action. The merit of this study is that by separating lymphocytes and incubating them in vitro, the medicinal effects can be examined separately on the vascular and lymphocyte sides. Fewer variables are easier to analyze.

Peyer&#8217;s patches (PPs) consist of hundreds of lymphoid follicles in the lamina propria of the small intestine. PPs are divided into follicles, the interfollicular region, and germinal centers located in the lower part of the follicles, where lymphocytes are stimulated by antigen presentation. There are no afferent lymphatic vessels, and the antigens invade the lamina propria from the intestinal lumen via the epithelial cell layer. The epithelial region covering lymphoid follicles is called the follicle-associated epithelium, within which specialized interspersed M cells uptake mucosal antigens. M cells take in antigens from the luminal side and antigens are then captured by dendritic cells and presented toward na&#239;ve lymphocytes that flow into PPs through the endothelium of high endothelial venules (HEVs)1. PPs play an important role in intestinal immunity and are related with the early stage of inflammation. Many molecular interactions involve the entrance of lymphocytes to secondary lymphoid organs (SLOs), including adhesion molecules, chemokines2,3, and sphingosine-1-phosphate4; thus, there are many expected therapeutic targets. Therefore, observing the lymphocyte dynamics within PPs enables us to catch a glimpse of the very early stage of inflammation and examine the usefulness of several promising drugs.
The method here focuses on the migration of lymphocytes in PPs, which includes several procedures (cannulation into the thoracic duct5 and collecting lymphocytes and long-term observation after the injection into collected lymphocytes). Since these procedures are complex and it was difficult to see exactly how each procedure was performed in previous reports, we mentioned here some tips to achieve a successful observation. For example, cannulation of the tubes into the thoracic duct was very difficult, and the initial success rate of cannulation was less than 50%. However, we improved the method and achieved a success rate exceeding 80%. We mentioned some other tips in this manuscript that are necessary for the successful observation to enable the quantitative evaluation of the transendothelial migration of lymphocytes under several conditions.
In previous reports, it was difficult to understand the three-dimensional changes over time, such as the intravenous injection of India ink to stain the vascular structure of PPs6, or the microscope being monofocal7. In recent years, an observational method using some photoconvertible fluorescence protein transgenic animals such as Kaede mice have clarified systematic cellular movements in vivo8. The other study clarified CD69 independent shutdown of lymphocyte egress from PPs9. We used confocal laser scanning microscopy (CLSM) because of its high analytical capability. Now we can easily obtain high-resolution images and use them to analyze lymphocyte dynamics.
In this report, we demonstrated a series of methods for evaluating lymphocyte migration in PPs. First, we showed refined methods of thoracic duct cannulation to collect lymphocytes. Second, we improved the observational methods in several ways to maintain objective organs whenever possible under microscopic observation, enabling us to obtain high-quality images for 3 hours. Third, we quantified the cellular movements of lymphocyte migration to evaluate the effects of some medications. These modified protocols will contribute to development of mucosal immunology evaluations.

<table><tbody><tr><td>A1R+</td><td>Nikon</td><td></td><td>Comfocal Laser Scanning Microscopy</td></tr><tr><td>Carboxyfluorescein diacetate succinimidyl ester</td><td>Thermo Fisher Scientific</td><td>C1157</td><td></td></tr><tr><td>Hoechest 33342</td><td>Thermo Fisher Scientific</td><td>H3570</td><td></td></tr><tr><td>Isoflurane</td><td>Wako Pure Chemical Industries</td><td>099-06571</td><td></td></tr><tr><td>RPMI 1640 medium</td><td>GIBCO</td><td>11875093</td><td></td></tr><tr><td>Texas Red&amp;ndash;dextran</td><td>Thermo Fisher Scientific</td><td>D1863</td><td></td></tr></tbody></table>

microscopic observation of lymphocyte dynamics in rat peyer's patches

Na&#239;ve lymphocytes recirculate from the blood to the lymphoid tissues under physiological condition and it is commonly recognized as an important phenomenon in the gut immunity. The stroma of secondary lymphoid organs, such as Peyer&#8217;s patches (PPs) or mesenteric lymph nodes, are where na&#239;ve lymphocytes sense antigens. Na&#239;ve lymphocytes circulate through the bloodstream to reach high endothelial venules, the portal of entry into PPs. Some immunomodulators are estimated to influence lymphocyte migration, but the precise evaluation of microcirculation dynamics is very difficult, and establishing a method to observe lymphocyte migration in vivo can contribute to the clarification of the precise mechanisms. We refined the method of collecting lymphocytes from the lymph duct and observing the detailed dynamics of gut-tropic lymphocytes in rat PPs. We chose confocal laser scanning microscopy to observe rat PPs in vivo and recorded it using time-lapse photography. We can now obtain clear images that can contribute to the analysis of lymphocyte dynamics.

Collecting lymphocytes from lymph 
To prepare the rat for thoracic duct cannulation, make an incision in the tense thoracic duct as shown in Figure 1 and then maintain the rat in a Bollman&#8217;s cage as shown in Figure 2.
When the lymphocytes are well collected, we can obtain about 20 mL/6 h lymph fluid containing about 107&#65374;108/mL lymphocytes. Of the TDLs, 70% express CD4, among which about 90% cells express CD62L, meaning the main composition of TDLs (&#62;60%) consists of na&#239;ve gut-tropic lymphocytes. Since the lymphocytes collected here are cells that specifically migrate to the intestinal tract, they are suitable for evaluating the state of migration in Peyer&#39;s patches.
Microscopic observation 
After injecting an adequate dose of fluorescent lymphocytes, mount the recipient rat&#8217;s intestine on a glass slide as shown in Figure 3. A microscopic image of PPs in the physiological condition is shown in Figure 4.
For the micro-observation, rats can be stably anesthetized under laparotomy for about 3 hours. Lymphocytes adhere to HEVs, roll, and migrate to the surrounding stroma, and then migrate to lymph capillaries. We quantified and evaluated the percentage of lymphocytes that migrated in the stroma within the observation period.
After the injection of an adequate dose of fluorescent lymphocytes, gut-tropic lymphocytes begin to adhere to HEVs and migrate across the endothelium into the stroma in about 1 hour. Most move within the stroma, while others migrate to lymph capillaries in about 2&#8211;3 hours. The well-developed fluorescence labeling ex vivo in this study resulted high intensity and it enables us to observe lymphocyte dynamics of very deep area. In addition, by using a different kind of fluorescence labelling, we can easily observe dynamics of different kind of lymphocyte simultaneously. This gut-tropic lymphocyte migration can be visually clarified for suppression by some immunomodulators such as FTY720 as shown in our previous report.
Observation of the microcirculatory system itself is extremely difficult, and it is desirable to establish an easy evaluating method. This study has also made it possible to find out where the action site of the drug is in the Peyer&#39;s patches. In particular, ex vivo administration of FTY720 to lymphocyte made it possible to limit the site of action only to lymphocytes but not to endothelium. Combined with the sorting of specific lymphocyte, more detailed results would be obtained.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61568/61568fig1.jpg" /> 
Figure 1: Procedure to expose the thoracic duct and perform cannulation. After cutting the peritoneum longitudinally, the thoracic duct can be exposed by moving the stomach cranially and dissecting the connective tissue on the dorsal right side of the abdominal aorta. The connective tissue around the diaphragm should be carefully dissected to prevent injury. The thoracic duct should be exposed as wide cranially as possible to create sufficient space for cannulation and then ligated by a 3-0 silk suture just under the diaphragm to stop the flow of lymph and retain the fluid caudally. Milky lymph fluid is visible through the lymph duct, where the catheter can be inserted. The thoracic duct of the rat should be slightly strained to ensure a smooth cannulation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61568/61568fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61568/61568fig2.jpg" /> 
Figure 2: An image of the entire setting to collect thoracic duct lymphocytes (TDLs). The rats are maintained in Bollman&#8217;s cages and TDLs are collected through the cannulation tube into each vial. The vials are set on ice and replaced every 12 hours to collect fresh lymph fluid. Sugar-laden saline is administered through a silicone tube using a syringe pump at a rate of around 3 mL/h to prevent dehydration. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61568/61568fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61568/61568fig3.jpg" /> 
Figure 3: The rat is put on the microscope&#8217;s stage connected to the anesthesia machine, and a fluorescent substance or fluorescently-labeled lymphocytes are injected from the venous catheter&#160;through the internal jugular vein (A). The observation area of the intestinal tract was gently fixed between two rolled cotton balls. The intestinal tract was fixed far from the trunk to avoid vibration by respiratory movements. Rolled cotton balls were soaked in phosphate buffered saline to prevent drying of the intestine during the observation period (B). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61568/61568fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61568/61568fig4.jpg" /> 
Figure 4: Microscopic image of Peyer&#8217;s patches in the physiological condition (A). The nuclei of vascular endothelial cells are stained blue. The blood plasma is red, and the blood flow is detected by the flow of unstained cells. Gut-tropic lymphocytes (stained green) adhering to high endothelial venules (B). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61568/61568fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Microscopic Observation of Lymphocyte Dynamics in Rat Peyer's Patches at JoVE.com

Microscopic Observation of Lymphocyte Dynamics in Rat Peyer's Patches

Here, we describe a precise method for collecting thoracic duct lymphocytes and observing the migration of gut-tropic lymphocytes in rat Peyer&#8217;s patches for 3 hours using time-lapse photography. This technique can clarify how the dynamics of lymphocytes are affected under inflammatory conditions.

Na&#239;ve lymphocytes recirculate from the blood to the lymphoid tissues under physiological condition and it is commonly recognized as an important ...

microscopic-observation-of-lymphocyte-dynamics-in-rat-peyer-s-patches

Nanyang Technological University

Research

JoVE Journal

Medicine

3.9K Views.  National Defense Medical College. Hier beschrijven we een nauwkeurige methode voor het verzamelen van thoracale kanaallymfocyten en het observeren van de migratie van darm-tropenlymfocyten in de patches van rat Peyer gedurende 3 uur met behulp van time-lapse fotografie. Deze techniek kan verduidelijken hoe de dynamiek van lymfocyten wordt beïnvloed onder inflammatoire omstandigheden.

Video: Microscopic Observation of Lymphocyte Dynamics in Rat Peyer's Patches

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes1, 2. To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer's patches. This antigen transcytosis is mediated by specialized epithelial M cells3, 4. M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane 5. Here, we present a method for the application of a mouse Peyer's patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described 6, 7. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer's patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer's patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer's patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.

Application of a Mouse Ligated Peyer&#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

M cells in a specialized follicle-associated epithelium covering Peyer&#x2019;s patches play an important role for the mucosal immunosurveillance in gut-associated lymphoid tissue. Here we described the evaluation method for bacterial transcytosis by M cells in vivo. This method provides a method to understand M-cell function in the immune system.

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

Immunology and Infection

Unraveling Key Players of Humoral Immunity: Advanced and Optimized Lymphocyte Isolation Protocol from Murine Peyer's Patches

In the gut mucosa, immune cells constitute a unique immunological entity, which promotes immune tolerance while concurrently conferring immune defense against pathogens. It is well established that Peyer&#39;s patches (PPs) have an essential role in the mucosal immune network by hosting several effector T and B cell subsets. A certain fraction of these effector cells, follicular T helper (TFH) and germinal center (GC) B cells are professionalized in the regulation of humoral immunity. Hence, the characterization of these cell subsets within PPs in terms of their differentiation program and functional properties can provide important information about mucosal immunity. To this end, an easily applicable, efficient and reproducible method of lymphocyte isolation from PPs would be valuable to researchers. In this study, we aimed to generate an effective method to isolate lymphocytes from mouse PPs with high cell yield. Our approach revealed that initial tissue processing such as the use of digestive reagents and tissue agitation, as well as cell staining conditions and selection of antibody panels, have great influence on the quality and identity of the isolated lymphocytes and on experimental outcomes.
Here, we describe a protocol enabling researchers to efficiently isolate lymphocyte populations from PPs allowing reproducible flow cytometry-based assessment of T and B cell subsets primarily focusing on&#160;TFH and GC B cell subsets.

In this study, we present a novel and effective protocol for the isolation of lymphocytes from Peyer&#39;s Patches (PPs), which can be subsequently used for in vivo and in vitro functional assays as well as flow cytometric studies of follicular T helper and germinal center B cells.

In the gut mucosa, immune cells constitute a unique immunological entity, which promotes immune tolerance while concurrently conferring immune defense ...

Cancer Research

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 ...

Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer's Patches

Peyer's patches (PPs) are integral components of the gut-associated lymphoid tissues (GALT) and play a central role in intestinal immunosurveillance and homeostasis. Particulate antigens and microbes in the intestinal lumen are continuously sampled by PP M cells in the follicle-associated epithelium (FAE) and transported to an underlying network of dendritic cells (DCs), macrophages, and lymphocytes. In this article, we describe protocols in which murine PPs are (i) dissociated into single cell suspensions and subjected to flow cytometry and (ii) prepared for cryosectioning and immunostaining. For flow cytometry, PPs are mechanically dissociated and then filtered through 70 &mu;m membranes to generate single cell suspensions free of epithelial cells and large debris. Starting with 20-25 PPs (from four mice), this quick and reproducible method yields a population of &gt;2.5 x 106 cells with &gt;90% cell viability. For cryosectioning, freshly isolated PPs are immersed in Optimal Cutting Temperature (OCT) medium, snap-frozen in liquid nitrogen, and then sectioned using a cryomicrotome. Tissue sections (5-12 &mu;m) are air-dried, fixed with acetone or methanol, and then subjected to immunolabeling.

There is an increasing interest in understanding the immunological functions of specific subpopulations of cells in Peyer's patches (PPs), the primary inductive sites of gut-associated lymphoid tissues. Here we outline parallel protocols for preparing PP single cell preparations for flow cytometric analysis and PP cryosections for immunostaining.

Peyer's  patches (PPs) are integral components of the gut-associated lymphoid tissues  (GALT) and play a central role in intestinal immunosurveillance and ...

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick tissue samples and organs in health and disease. By controlling the scanning pattern in multi-photon microscopy and applying appropriate numerical algorithms, we developed a striped-illumination approach, which enabled us to achieve 3-fold better axial resolution and improved signal-to-noise ratio, i.e. contrast, in more than 100 &#181;m tissue depth within highly scattering tissue of lymphoid organs as compared to standard multi-photon microscopy. The acquisition speed as well as photobleaching and photodamage effects were similar to standard photo-multiplier-based technique, whereas the imaging depth was slightly lower due to the use of field detectors. By using the striped-illumination approach, we are able to observe the dynamics of immune complex deposits on secondary follicular dendritic cells &#8211; on the level of a few protein molecules in germinal centers.

High-resolution intravital imaging with enhanced contrast up to 120 &#181;m depth in lymph nodes of adult mice is achieved by spatially modulating the excitation pattern of a multi-focal two-photon microscope. In 100 &#181;m depth we measured resolutions of 487 nm (lateral) and 551 nm (axial), thus circumventing scattering and diffraction limits.

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick ...

An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics

Adaptive immunity is regulated by dynamic interactions between T cells and antigen presenting cells (&#39;APCs&#39;) referred to as &#39;immunological synapses&#39;. Within these intimate cell-cell interfaces discrete sub-cellular clusters of MHC/Ag-TCR, F-actin, adhesion and signaling molecules form and remodel rapidly. These dynamics are thought to be critical determinants of both the efficiency and quality of the immune responses that develop and therefore of protective versus pathologic immunity. Current understanding of immunological synapses with physiologic APCs is limited by the inadequacy of the obtainable imaging resolution. Though artificial substrate models (e.g., planar lipid bilayers) offer excellent resolution and have been extremely valuable tools, they are inherently non-physiologic and oversimplified. Vascular and lymphatic endothelial cells have emerged as an important peripheral tissue (or stromal) compartment of &#39;semi-professional APCs&#39;. These APCs (which express most of the molecular machinery of professional APCs) have the unique feature of forming virtually planar cell surface and are readily transfectable (e.g., with fluorescent protein reporters). Herein a basic approach to implement endothelial cells as a novel and physiologic &#39;planar cellular APC model&#39; for improved imaging and interrogation of fundamental antigenic signaling processes will be described.

Adaptive immunity is controlled by dynamic 'immunological synapses' formed between T cells and antigen presenting cells. This protocol describes methods for investigating endothelial cells both as understudied physiologic APCs and as a novel type of 'planar cellular APC model'.

Adaptive immunity is regulated by dynamic interactions between T cells and antigen presenting cells (&#39;APCs&#39;) referred to as &#39;immunological ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...

Traction Force Microscopy to Study B Lymphocyte Activation

Traction force microscopy (TFM) enables the measurement of forces produced by a cell on a substrate. This technique infers traction force measurements from an experimentally observed displacement field produced by a cell pulling on an elastic substrate. Here, we adapted TFM to investigate the spatial and temporal structure of the force field exerted by B cells when activated by antigen engagement of the B cell receptor. Gel rigidity, bead density, and protein functionalization must be optimized for the study of relatively small cells (~ 6 &#181;m) that interact with, and respond specifically to ligands for cell surface receptors.

Here we present a protocol used to perform traction force microscopy experiments on B cells. We describe the preparation of soft polyacrylamide gels and their functionalization, as well as data acquisition at the microscope and a summary of data analysis.

Traction force microscopy (TFM) enables the measurement of forces produced by a cell on a substrate. This technique infers traction force measurements ...

Bioengineering

Quantifying Leukocyte Egress via Lymphatic Vessels from Murine Skin and Tumors

Leukocyte egress from peripheral tissues to draining lymph nodes is not only critical for immune surveillance and initiation but also contributes to the resolution of peripheral tissue responses. While a variety of methods are used to quantify leukocyte egress from non-lymphoid, peripheral tissues, the cellular and molecular mechanisms that govern context-dependent egress remain poorly understood. Here, we describe the use of in situ photoconversion for quantitative analysis of leukocyte egress from murine skin and tumors. Photoconversion allows for the direct labeling of leukocytes resident within cutaneous tissue. Though skin exposure to violet light induces local inflammatory responses characterized by leukocyte infiltrates and vascular leakiness, in a head-to-head comparison with transdermal application of fluorescent tracers, photoconversion specifically labeled migratory dendritic cell populations and simultaneously enabled the quantification of myeloid and lymphoid egress from cutaneous microenvironments and tumors. The mechanisms of leukocyte egress remain a missing component in our understanding of intratumoral leukocyte complexity, and thus the application of the tools described herein will provide unique insight into the dynamics of tumor immune microenvironments both at steady state and in response to therapy.

Here, we demonstrate the methods for in vivo quantification of leukocyte egress from na&#239;ve, inflamed, and malignant murine skin. We perform a head-to-head comparison of two models: transdermal FITC application and in situ photoconversion. Furthermore, we demonstrate the utility of photoconversion for tracking leukocyte egress from cutaneous tumors.

Leukocyte egress from peripheral tissues to draining lymph nodes is not only critical for immune surveillance and initiation but also contributes to the ...

Biology

Isolation of Lymphocytes from a Mouse Peyer&#39;s Patch

Source: Yazicioglu, Y. F., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/58490">Unraveling Key Players of Humoral Immunity: Advanced and Optimized Lymphocyte Isolation Protocol from Murine Peyer&#39;s Patches.</a> J. Vis. Exp. (2018)
In this video, we demonstrate the isolation of lymphocytes from a harvested mouse Peyer&#39;s patch. The isolated lymphocytes can be used for further analysis.

Source: Yazicioglu, Y. F., et al. Unraveling Key Players of Humoral Immunity: Advanced and Optimized Lymphocyte Isolation Protocol from Murine Peyer&#39;s ...

Microscopic Observation of Lymphocyte Dynamics in Rat Peyer's Patches

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