The authors thank Ava Zardynezhad for proofreading of the manuscript. This work is supported by the Canadian Institute of Health Research (CIHR, PJT-156035), and the Canada Foundation for Innovation for SL (32930), and by the National Natural Science Foundation of China for Yujia Lin (81901576).

All animal work needs to be approved by institutional and governmental ethics and animal handling committee.&#160; This is a non-survival surgery.&#160;
1. Preparation of materials
<ol>
	<li>Prepare 100 mL of 70% ethanol by mixing 70 mL of 100% ethanol with 30 mL of sterile water. Autoclave all surgical tools before surgery and keep the tools in 70% ethanol before and during the surgery to maintain sterilization.</li>
	<li>Prepare an injection apparatus.
	<ol>
		<li>Cut ~30 cm of polyethylene tubing (0.28 mm in diameter). Connect the tip of a 30 G x &#189; needle (needle A) to one end of polyethylene tubing. Carefully dislodge another 30 G x &#189; needle (needle B) and connect the broken side to the other end of polyethylene tubing.</li>
		<li>Attach needle A to a 1 mL tuberculin syringe. 
		NOTE: For this polyethylene tubing, 1.6 cm of fluid in the tubing corresponds to 1 &#956;L20.</li>
	</ol>
	</li>
	<li>Prepare a 10:1 ketamine/xylazine mixture (10 mg/mL ketamine and 1 mg/mL xylazine) in saline (bacteriostatic 0.9% [w/v] sodium chloride). Prepare the solution freshly before use.</li>
</ol>
2. Preparation of the animal for surgery
NOTE: Use mice aged 6&#8722;10 weeks. Both female and male mice can be used. In this study, 6&#8722;10-week-old, C57BL/6 female mice were used. This method can be adapted for other strains of mice.
<ol>
	<li>Anesthetize the mouse by injecting 250 &#956;L of the ketamine/xylazine mixture intraperitoneally. Wait until the mouse is completely asleep. Ensure mouse does not react to a toe pinch to detect full anesthetization.</li>
	<li>Shave fur around the legs with hair clippers.</li>
	<li>Apply the depilatory cream around the leg and wait for 5 min. Wipe off the residual fur and the depilatory cream using a moist tissue and clean the leg with sterile water. Spray 70% ethanol around the leg to sterilize the operating area. The ethanol is restricted to the incision site.&#160;</li>
</ol>
3. Surgical suturing of afferent lymphatic vessels
NOTE: The right leg is sutured, and the left leg is used as the sham control. The lymphatic suture protocol (steps 3.1&#8722;3.8) takes 20&#8722;30 min.
<ol>
	<li>Keep mouse at a prone position and fix it with surgical tape to expose the operation area on the right leg.</li>
	<li>Intradermally inject 5 &#181;L of 1% Evans blue dye or 9 cm of the fluid of the injection apparatus tubing into the footpad. Gently massage the footpad to help Evans blue enter the lymphatic vessels. 
	NOTE: The insulin syringe is not easy to control for small volume injection. The volume can be controlled more accurately using the injection apparatus. Lymphatic vessels are visualized by blue dye under the skin. With extensive training, both afferent lymphatic vessels can be seen with the naked eye as transparent vessels in the adipose tissue, parallel to the Saphenous artery. With extensive training, it is possible to suture the vessels without injecting Evans Blue dye in cases where there are concerns of potential disturbances from the dye.</li>
	<li>Under a dissecting microscope, choose an incision site 5 mm from the bottom edge of the popliteal fossa. Make a small incision (~5 mm) in the skin with scissors. Using fine operation forceps, stretch the incision, and expose the collecting lymphatic vessels (Figure 1A). 
	NOTE: If necessary, a small skin fragment can be removed to expose the lymphatic vessels.</li>
	<li>Identify both afferent lymphatic vessels leading to the pLNs under the dissecting microscope (Figure 1B). 
	NOTE: There are two afferent lymphatic vessels from the footpad to the pLN. Both need to be sutured to block lymph flow completely.</li>
	<li>Using a needle holder, cautiously insert the suture needle (0.7 metric or smaller) between the afferent lymphatic vessel and the Saphenous artery and pull the needle gently out around the afferent lymphatic vessel. Gently pull the suture string and leave about 2 cm of the suture string behind. Use the needle holder to help tie the string tightly to suture one lymphatic vessel with a surgeon&#8217;s knot (Figure 1C). 
	NOTE: The tissue underneath the incision may dry out with prolonged exposure to air. Making the incision as small as possible and performing the suture quickly (i.e., within 5 min) will prevent the tissue from drying out. Maintain the tissue moisture by applying a small volume of saline with a cotton swab.</li>
	<li>Gently massage the footpad to ensure no Evans Blue dye passes the suture site and then cut the excess string with scissors.</li>
	<li>Perform the same suture steps (i.e., steps 3.5 and 3.6) on the other afferent lymphatic vessel (Figure 1D). Close the skin incision with the same suture that was used to suture the vessels in step 3.5 (Figure 1E).</li>
	<li>For the sham control, intradermally inject 5 &#181;L of 1% Evans blue dye at the left footpad and massage the footpad to visualize the lymphatic vessels. Open the skin with an excision and then close the wound without suturing the vessel (Figure 1F).</li>
	<li>Optionally, monitor the operated mice for 2&#8722;4 h. The suture side of the leg should show edema with Evans Blue spread to the thigh, while the control leg will show restricted Evans blue dye in the footpad.&#160;If mice&#160;awaken, an additional ketamine/xylazine&#160;mix will be injected to keep anesthetized until euthanasia.</li>
</ol>
4. Tracking of the lymph flow
<ol>
	<li>Immediately after the surgery, intradermally inject 10 &#181;L of 2% fluorescein isothiocyanate (FITC) in the footpad of both the control and the lymphatic sutured leg.</li>
	<li>Euthanize the mice with 400 &#956;L of ketamine/xylazine mixture and perform cervical dislocation when the mice are fully anesthetized.</li>
	<li>Collect pLNs from the popliteal fossa and carefully remove the perinodal adipose tissue around the pLNs under the dissection microscope at 2, 6, and 12 h after FITC injection.</li>
	<li>Embed the pLNs with the medullary sinus area facing to the side of the cryomold in optimal cutting temperature (OCT) compound (Figure 1G,H).</li>
	<li>Prepare 20 &#181;m frozen sections using a cryotome.</li>
	<li>Image the cryosections under a confocal microscope to determine FITC distribution.</li>
</ol>

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The authors have no conflicts of interest to disclose.

Blocking lymph flow will have broad applications in manipulating antigen delivery to the LN in healthy and diseased conditions. It is possible to use this method to control the timing of antigen delivery in order to study how continuous lymph flow regulates immune response in draining LNs. This method of lymph flow interruption can also be used to study how lymph impacts cell compartmentalization, cell activation, cell migration, and cell-cell interactions in the LN.
Mice specifically expressi...

The spatial organization of the lymphatic system provides structural and functional support to efficiently remove extracellular fluid and transport antigens and antigen-presenting cells (APCs) to the draining LNs. The initial lymphatic vessels (also named lymphatic capillaries) are highly permeable due to their discontinuous intercellular junctions, which facilitate the effective collection of fluids, cells, and other materials from surrounding extracellular spaces1. The initial lymphatic vessels merge into collecting lymphatic vessels, which have tight intercellular junctions, a continuous basement membrane, and lymphatic muscle coverage. Collecting lymphatic vessels are responsible for transporting collected lymph to the draining LNs and eventually returning lymph to the circulation2,3. The collecting lymphatic vessels that propel lymph into the draining LN are the afferent lymphatic vessels4,5,6,7. Obstruction of afferent lymphatic vessels can block lymph flow into the LNs, which is a useful technique when studying the function of lymph flow.
Previous studies have shown that lymph flow plays a significant role in transporting antigens and APCs, as well as maintaining LN homeostasis. It is well understood that tissue-derived APCs, typically activated migrating dendritic cells (DCs), travel through the afferent lymphatic vessels to the LN to activate T cells8. The idea that free-form antigens, such as microbes or soluble antigens, passively flow with lymph to the LN to activate LN-resident APCs has been gaining acceptance in the past decade9,10,11,12. Free-form antigens traveling with lymph take minutes after the infection to travel to the LN, and the LN-resident cell activation may occur within 20 min after the stimulation. This is much faster than the activation of migrating DCs, which takes more than 8 h to enter the draining LN9. Besides transporting antigens to initiate immune protection, lymph also carries cytokines and DCs to the LN to maintain its microenvironment, and to support immune cell homeostasis13,14. Previously, blocking lymph flow by suturing the afferent lymphatic vessels demonstrated that lymph is required to maintain the HEV phenotype required for supporting homeostatic T cell and B cell homing to the LN15,16,17. CCL21 is a critical chemokine that directs DC and T cell positioning in the LN8,18. Blocking lymph flow interrupts CCL21 expression in the LN and potentially interrupts DC and T cell positioning and/or interaction in the LN19. Thus, blocking lymph flow can directly or indirectly abrogate antigen/DC access to the draining LN by disrupting the LN microenvironment that regulates immune responses in the LN. To better investigate the function of lymph flow, an experimental protocol is presented (Figure 1) to block lymph flow in mice by suturing the afferent lymphatic vessels from the footpad to the pLN. This method can be an important technique for future studies on lymphatic function in healthy and diseased conditions.

<table>
	<tbody>
		<tr>
			<td>0.9% Sodium Chloride Saline</td>
			<td>Baxter</td>
			<td>JB1323</td>
			<td></td>
		</tr>
		<tr>
			<td>100% ethanol</td>
			<td>Greenfield Global</td>
			<td></td>
			<td>University of Calgary distribution services UN1170.</td>
		</tr>
		<tr>
			<td>Depilatory cream</td>
			<td>Nair</td>
			<td></td>
			<td>Nair Sensitive Formula Hair Removal Cr&#232;me with Sweet Almond Oil and Baby Oil, 200-ml. Or similar product.</td>
		</tr>
		<tr>
			<td>Evans Blue dye</td>
			<td>Sigma Life Science</td>
			<td>E2129-10G</td>
			<td>For 1 ml of Evans blue dye, add 0.1g Evans blue to 10 ml PBS. The Evens Blue solution will be filtered through 0.22 mm filters and kept sterile in 1ml aliquots.</td>
		</tr>
		<tr>
			<td>Fluorescein isothiocyanate isomer I (FITC)</td>
			<td>Sigma Life Science</td>
			<td>F7250-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps Dumont #3</td>
			<td>WPI</td>
			<td>500337</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps Dumont #5</td>
			<td>WPI</td>
			<td>500233</td>
			<td></td>
		</tr>
		<tr>
			<td>Injection apparatus</td>
			<td></td>
			<td></td>
			<td>Connect one end of polyethylene tubing to 30G &#215; &#189; needle. Attach a 1ml TB syringe to the needle. Dislodge needle shaft from another 30G &#215; &#189; needle. Insert the blunt end of the 30G &#215; &#189; needle shaft into the other end of the tubing. The inside diameter of this tubing is 0.28mm. Thus, 1.6 cm of fluid in the tubing is 1 &#956;l.</td>
		</tr>
		<tr>
			<td>Insulin syringe</td>
			<td>Becton Dickinson and Company (BD)</td>
			<td>329461</td>
			<td></td>
		</tr>
		<tr>
			<td>IRIS Forcep straight</td>
			<td>WPI</td>
			<td>15914</td>
			<td></td>
		</tr>
		<tr>
			<td>IRIS scissors</td>
			<td>WPI</td>
			<td>14218-G</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Narketan</td>
			<td>DIN 02374994</td>
			<td>The suppliers of Ketamine and Xylazine are usually under institutional and governmental regulation.</td>
		</tr>
		<tr>
			<td>Needles (26Gx3/8)</td>
			<td>Becton Dickinson and Company (BD)</td>
			<td>305110</td>
			<td></td>
		</tr>
		<tr>
			<td>Needles (30Gx1/2)</td>
			<td>Becton Dickinson and Company (BD)</td>
			<td>305106</td>
			<td></td>
		</tr>
		<tr>
			<td>Paton Needle Holder</td>
			<td>ROBOZ</td>
			<td>RS6403</td>
			<td>Straight, Without Lock; Serrated</td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline (PBS)</td>
			<td>Sigma Life Science</td>
			<td>P4417-100TAB</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene tubing</td>
			<td>Becton Dickinson and Company (BD)</td>
			<td>427401</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical tape (1.25cmx9.1m )</td>
			<td>Transpore</td>
			<td>1527-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical tape (2.5cmx9.1m )</td>
			<td>Transpore</td>
			<td>1527-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture</td>
			<td>Davis and Geck CYANAMID Canada</td>
			<td>11/04</td>
			<td>0.7 metric monofilament polypropylene</td>
		</tr>
		<tr>
			<td>Syringe (1ml)</td>
			<td>Becton Dickinson and Company (BD)</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>VANNAS scissors</td>
			<td>World Precision Instruments (WPI)</td>
			<td>14122-G</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Rompun</td>
			<td>DIN02169606</td>
			<td>The suppliers of Ketamine and Xylazine are usually under institutional and governmental regulation.</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Olympus</td>
			<td></td>
			<td>Olympus S261 (522-STS OH141791) with light source: Olympus Highlight 3100</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Leica</td>
			<td></td>
			<td>SP8</td>
		</tr>
	</tbody>
</table>

blocking lymph flow by suturing afferent lymphatic vessels in mice

Lymphatic vessels are critical in maintaining tissue fluid balance and optimizing immune protection by transporting antigens, cytokines, and cells to draining lymph nodes (LNs). Interruption of lymph flow is an important method when studying the function of lymphatic vessels. The afferent lymphatic vessels from the murine footpad to the popliteal lymph nodes (pLNs) are well-defined as the only routes for lymph drainage into the pLNs. Suturing these afferent lymphatic vessels can selectively prevent lymph flow to the pLNs. This method allows for interference in lymph flow with minimal damage to the lymphatic endothelial cells in the draining pLN, the afferent lymphatic vessels, as well as other lymphatic vessels around the area. This method has been used to study how lymph impacts high endothelial venules (HEV) and chemokine expression in the LN, and how lymph flows through the adipose tissue surrounding the LN in the absence of functional lymphatic vessels. With the growing recognition of the importance of lymphatic function, this method will have broader applications to further unravel the function of lymphatic vessels in regulating the LN microenvironment and immune responses.

Lymphatic vessel suture has been used in previous studies15,16,17,19, where it served as an important tool to study the function of lymph flow before the molecular biology of lymphatic vessels was better understood. Blocking lymph flow interrupts LN homeostasis, which leads to HEVs losing the critical gene expression needed for optimal lymphocyte homing to the LN15,...

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Blocking Lymph Flow by Suturing Afferent Lymphatic Vessels in Mice

A protocol to block lymph flow by surgical suturing of afferent lymphatic vessels is presented.

Lymphatic vessels are critical in maintaining tissue fluid balance and optimizing immune protection by transporting antigens, cytokines, and cells to ...

blocking-lymph-flow-by-suturing-afferent-lymphatic-vessels-in-mice

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JoVE Journal

Immunology and Infection

5.3K Views.  Harbin Medical University. A protocol to block lymph flow by surgical suturing of afferent lymphatic vessels is presented.

Video: Blocking Lymph Flow by Suturing Afferent Lymphatic Vessels in Mice

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, ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

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

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

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

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

Digestion of the Murine Liver for a Flow Cytometric Analysis of Lymphatic Endothelial Cells

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the lymph nodes where cellular debris and antigens can be surveyed. We are very interested in understanding how the lymphatic vasculature might be involved in inflammation and immune cell function within the liver. However, very little has been published establishing digestion protocols for the isolation of lymphatic endothelial cells (LECs) from the liver or specific markers that can be used to evaluate liver LECs on a per cell basis. Therefore, we optimized a method for the digestion and staining of the liver in order to evaluate the LEC population in the liver. We are confident that the method outlined here will be useful for the identification and isolation of LECs from the liver and will strengthen our understanding of how LECs respond to the liver microenvironment.

The goal of this protocol is to identify lymphatic endothelial cell populations within the liver using described markers. We utilize collagenase IV and DNase and a gentle mincing of tissue, combined with flow cytometry, to identify a distinct population of lymphatic endothelial cells.

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the ...

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.

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, ...

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are cleared from the human lung alveoli by being phagocytosed by innate monocytes and/or neutrophils. This protocol offers a fast and reliable measurement of phagocytosis by flow cytometry using fluorescein isothiocyanate (FITC)-labeled conidia for co-incubation with human leukocytes and subsequent counterstaining with an anti-FITC antibody to allow discrimination of internalized and cell-adherent conidia. Major advantages of this protocol are its rapidness, the possibility to combine the assay with cytometric analysis of other cell markers of interest, the simultaneous analysis of monocytes and neutrophils from a single sample and its applicability to other cell wall-bearing fungi or bacteria. Determination of percentages of phagocytosing leukocytes provides a means to microbiologists for evaluating virulence of a pathogen or for comparing pathogen wildtypes and mutants as well as to immunologists for investigating human leukocyte capabilities to combat pathogens.

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

This protocol provides a fast and reliable method to quantitatively measure phagocytosis of Aspergillus fumigatus conidia by human primary phagocytes using flow cytometry and to discriminate phagocytosis of conidia from mere adhesion to leukocytes.

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are ...

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of UTI in male and female mice have illustrated the procedures for bacterial inoculation and enumeration in urine and tissues. During an initial bladder infection in C57BL/6 mice, UPEC establish latent reservoirs inside bladder epithelial cells that persist following clearance of UPEC bacteriuria. This model builds on these studies to examine rUTI caused by the emergence of UPEC from within latent bladder reservoirs. The urogenital bacterium Gardnerella vaginalis is used as the trigger of rUTI in this model because it is frequently present in the urogenital tracts of women, especially in the context of vaginal dysbiosis that has been associated with UTI. In addition, a method for in situ bladder fixation followed by scanning electron microscopy (SEM) analysis of bladder tissue is also described, with potential application to other studies involving the bladder.

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

A mouse model of uropathogenic E. coli (UPEC) transurethral inoculation to establish latent intracellular bladder reservoirs and subsequent bladder exposure to G. vaginalis to induce recurrent UPEC UTI is demonstrated. Also demonstrated are the enumeration of bacteria, urine cytology, and in situ bladder fixation and processing for scanning electron microscopy.

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of ...

﻿Autofluorescence Blocking and Antigen Retrieval in Tissue Samples

Autofluorescence Blocking and Antigen Retrieval in Tissue Samples  

Real-Time Analysis of Calcium and Contractility in Isolated Lymph Vessels

Real-time Evaluation of Absolute, Cytosolic, Free Ca2+ and Corresponding Contractility in Isolated, Pressurized Lymph Vessels

The lymphatic vasculature, now often referred to as &#34;the third circulation,&#34; is located in many vital organ systems. A principal mechanical function of the lymphatic vasculature is to return fluid from extracellular spaces back to the central venous ducts. Lymph transport is mediated by spontaneous rhythmic contractions of lymph vessels (LVs). LV contractions are largely regulated by the cyclic rise and fall of cytosolic, free calcium ([Ca2+]i).
This paper presents a method to concurrently calculate changes in absolute concentrations of [Ca2+]i and vessel contractility/rhythmicity in real time in isolated, pressurized LVs. Using isolated rat mesenteric LVs, we studied changes in [Ca2+]i and contractility/rhythmicity in response to drug addition. Isolated LVs were loaded with the ratiometric Ca2+-sensing indicator Fura-2AM, and video microscopy coupled with edge-detection software was used to capture [Ca2+]i&#160;and diameter measurements continuously in real time.
The Fura-2AM signal from each LV was calibrated to the minimum and maximum signal for each vessel and used to calculate absolute [Ca2+]i. Diameter measurements were used to calculate contractile parameters (amplitude, end diastolic diameter, end systolic diameter, calculated flow) and rhythmicity (frequency, contraction time, relaxation time) and correlated with absolute [Ca2+]i measurements.

Author Spotlight: Innovative Methods in Lymphedema and Hypertension Research

This protocol describes a method to simultaneously measure cytosolic, free calcium [Ca2+]i and vessel diameter in contracting lymph vessels in real time and then calculate absolute Ca2+ concentrations as well as contractility/rhythmicity parameters. This protocol can be used to study Ca2+ and contractile dynamics across a variety of experimental conditions.

The lymphatic vasculature, now often referred to as &#34;the third circulation,&#34; is located in many vital organ systems. A principal mechanical ...

Quantifying Pyroptosis in Infectious Bursal Disease Virus-Infected DF-1 Cells

Examination of Pyroptosis by Flow Cytometry

Pyroptosis is an inflammatory type of programmed cell death predominantly driven by the formation of plasma membrane pores by the N-terminus generated from the cleaved Gasdermin (GSDM) family proteins. Examination of membrane-attached GSDM-NT by Western Blot is the most commonly used method for evaluating pyroptosis. However, it is difficult to differentiate cells with pyroptosis from other forms of cell death using this method. In this study, Infectious Bursal Disease Virus (IBDV)-infected DF-1 cells were employed as a model to quantify the proportion of cells undergoing pyroptosis by flow cytometry, utilizing specific antibodies against the N-terminal fragment of chicken GSDME (chGSDME-NT) and propidium iodide (PI) staining. The chGSDME-NT-positive cells were readily detectable by flow cytometry using Alexa Fluor 647-labeled anti-chGSDME-NT antibodies. Moreover, the proportion of chGSDME-NT/PI double-positive cells in IBDV-infected cells (around 33%) was significantly greater than in mock-infected controls (P &#60; 0.001). These findings indicate that examination of membrane-bound chGSDME-NT by flow cytometry is an effective approach for determining pyroptotic cells among cells undergoing cell death.

Author Spotlight: Flow Cytometric Determination of Pyroptosis in Avian Cells

This article describes the identification of pyroptotic cells using flow cytometry after dual staining with antibodies against the N-terminal fragment of chicken GSDME (chGSDME-NT) and propidium iodide (PI).

Pyroptosis is an inflammatory type of programmed cell death predominantly driven by the formation of plasma membrane pores by the N-terminus generated ...