1. Before the process, prepare and gather the following items
<ol>
	<li>Please refer to the image in Figure 1a) and 1b) for building a wooden platform to restrain the mouse during the procedure. </li>
	<li>Digestion mix for lymph nodes - HBSS + 1.25mg/ml Collagenase TypeIV or 2.5 mg/ml Collagenase D</li>
	<li>Digestion mix for lungs - HBSS + 1 mg/ml Collagenase </li>
	<li>Dilute latex beads (1:20) in PBS for intraperitoneal injection to visualize the LDLNs </li>
</ol>
Note: LDLNs are small and hard to find. To learn how to find them, inject 200&mu;l of the 1:20 diluted red fluorescent latex 
beads i.p into the mouse. The peritoneum drains to these LDLNs8. 24 hpi, expose the thoracic cavity of the mouse as described above. The LDLNs will appears pink from the beads that drained from the peritoneal cavity. 
<ol start="5">
	<li>Red Blood Cell (RBC) Lysis Buffer - 0.15M NH4Cl, 10mM KHCO3, 0.1 mM Na2EDTA in 1L dH20. Adjust pH to 7.2-7.4 and store at room temperature.</li>
	<li>70&mu;m or 100&mu;m cell strainer </li>
	<li>Stock solution of 0.5M EDTA, pH = 8.0 </li>
	<li>HBSS without CaCl2 and MgCl2</li>
	<li>Scissors and blunt-ended forceps </li>
	<li>Laryngoscope </li>
	<li>Gavage needle (22 gauge) bent at an angle (Figure 2)</li>
	<li>Styrofoam board and pins for spreading the mouse</li>
	<li>Top pan balance</li>
	<li>Aniline Blue Solution - 10mg/ml in H2O</li>
</ol>
Note: We will be using this solution for demonstration of i.t injection technique. 
2. Preparation and administration of mouse anesthetic 
<ol>
	<li>Prepare a mixture of Xylazine and Ketamine in sterile PBS so that the final concentrations are 2mg/ml and 10mg/ml respectively.</li>
	<li>Weigh the mouse on a top pan balance. </li>
	<li>Calculate the dose required for each mouse. Dose at Xylazine (2-4 mg/kg) and Ketamine (70-80 mg/kg) body weight using appropriate volume of the prepared mixture and inject intaperitoneally. Each mouse strain may require optimization of anesthetic. Mice receiving too much anesthetic may have a reduced respiration rate that could be fatal after inoculum is instilled into the lungs. </li>
	<li>After 3-5 minutes, observe the mouse for the effects of the anesthetic. Check for the following signs to confirm that the mouse is fully anesthetized:
		<ol>
			<li>The breathing rate should slow down.</li>
			<li>The mouse should not stretch its arm when picked up by the neck.</li>
			<li>There should not be any response when the hind limbs are stimulated. 
			If these criteria are not met, wait for a few minutes and check again before proceeding to the next step. </li>
		</ol>
	</li>
</ol>
3. Intratracheal injection 
<ol>
	<li>Prepare the innoculum in 50&mu;l saline. Fill a 1ml syringe fitted with a sterile bent gavage needle with the innoculum. The syringe should be loaded with a 
	100&mu;l air pocket behind the innoculum to ensure that all of the fluid is instilled into the lung. </li>
	<li>Place the mouse on the angled wooden platform hanging by its incisors on the wire and gently restrain it in place with a piece of ribbon. </li>
	<li>Turn on the laryngoscope with your left hand (if you are right handed) and grab a pair of blunt ended forceps. Use the tip of laryngoscope and the forceps to gently pry open the mouth.</li>
	<li>With the forceps, pull the tongue out and hold it to the side. Guide the laryngoscope blade towards the back of the mouth. Keep the laryngoscope pressed down very gently at a 90&deg; angle until you see the opening of the trachea. Hold the laryngoscope in place. Care should be taken when pulling the tongue from the mouth and this should be done gently so no tissue damages occurs. </li>
	<li>With your other hand, take the 1ml syringe containing the innoculum, fitted with the bent gavage needle with your right hands and insert the needle into the trachea until the bend in the needle is by the front incisors. Push the plunger evenly to deliver the innoculum. The innoculum should not bubble. Pull the needle out of the trachea as soon as possible. Mice will suffocate and die if the trachea is blocked for too long. Hold the mouse upright for a few seconds to allow innoculum to be inhaled into the lungs. </li>
	<li>Take the mouse out of the platform and place it near a heating lamp. Do not leave mice under direct heat for extended periods of time. The mouse should be fully awake within 30 min after the procedure (recovery time may vary based on strains). Mice should be observed diligently during this time and should not be allowed to get too hot or too cold, this could also put the mice into respiratory distress leading to death.</li>
</ol>
4. Harvesting and processing Lung draining lymph nodes and lungs for flow cytometry 
<ol>
	<li>Sacrifice mice using the desired method as per the AVMA euthanasia guidelines. This may vary depending upon individual harvest outcomes. Administration of a lethal does of sodium pentobarbital is an example of an appropriate euthanasia procedure. We have not seen negative pulmonary effects of euthanasia by CO2.</li>
	<li>Pin the arms and legs on a dissection board. Make an incision along the middle of the ventral side with scissors. Gently pull the skin and expose the thoracic wall muscles. </li>
	<li>Make incisions through the thoracic wall muscles to expose the abdominal organs.</li>
	<li>Hold the tip of sternum with the forceps and puncture the diaphragm. Cut the diaphragm along the sides of the rib cage. </li>
	<li>With the scissors parallel to the dissection board surface, cut through the thoracic cavity along the dorso-ventral line on both sides. </li>
	<li>Slightly lift the rib cage up. On the right side of the heart, just underneath the thymus, look for two lymph nodes. A slightly larger third lymph node is located underneath a small arterial blood vessel that runs perpendicular to the trachea. Harvest lymph nodes into 1 ml of lymph node digestion mix.</li>
	<li>After harvesting the lymph nodes, hold the heart with a pair of forceps and grab the lungs with another. Harvest lungs into 3ml of ice cold PBS in a 6-well plate. </li>
	<li>Separate the lobes of the lungs, aspirate PBS and chop the lobes into small pieces using forceps and scissors. Add 3ml of the digestion mix for lungs. Keep the 6-well plate on ice during this step. </li>
	<li>Incubate the lymph nodes for 25-30 min and lungs for 30 min at 37&deg;C. Add EDTA to a final concentration of 10mM to each of the digestion mixtures to stop the reaction and place on ice. </li>
	<li>Using a 3ml syringe, pass pieces of lungs through an 18GA needle 3 times. Mash the pieces of the lungs and the LNs through 70-100&mu;m cell strainers using 
	the back end of a 1ml syringe plunger. Wash the cell strainers with 10ml of HBSS without Ca2+ and Mg2+. Centrifuge at 500 x g for 5 min. </li>
	<li>Resuspend the pellet in 5 ml of RBC lysis buffer and incubate for 3 min at room temperature. Add 10ml of HBSS without Ca2+ and Mg2+ to quench lysis. 
	Centrifuge at 500 x g for 5 min. Bring up the lung and LDLN single cell suspensions to 10ml and 5ml respectively. Count live cells using a hemacytometer 
	by trypan blue exclusion. Cells are now ready to stain for flow cytometry. 1 lobe of the lung might be sufficient for flow cytometry. Other lobes can be 
	used for other experimental questions within the same mouse such as homogenates for cytokines or tissue for histology.</li>
</ol>
5. Representative Results: 
LDCs and alveolar macrophages in the lungs acquire antigen from the alveolar spaces of the lungs immediately following i.t injection. 
Figure 3 shows percentage of CD11c+ (expressed on LDCs and alveolar macrophages) cells that are positive for fluorescently labeled Bacillus anthracis spores, 
30 min post i.t injection. At this time, ~15.9% of CD45+CD11c+ cells in the lung single cell suspension harbored Bacillus anthracis spores (Figure 3, lower
 panels). For further characterization of CD11c+ cells in the lungs, refer to Kim and Braciale4. Fluorescently labeled beads are routinely used to assess 
trafficking of particulate antigens to LDLNs9. To assess this transport from lungs to LDLNs, fluorescent latex beads were injected i.t with LPS and LDLNs 
were analyzed at 24 hpi. About 4% of CD11chi cells in the LDLNs harbored fluorescent beads at 24hpi (Figure 4). These representative experiments show that 
this protocol can be efficiently used to track cells that acquire inhaled particulate antigens in the lungs and subsequently traffic them to the LDLNs.

<img src="/files/ftp_upload/2702/2702fig1.jpg" alt="Figure 1"/> 
Figure 1. Wooden platform for restraining mice during i.t injection. A wooden block was cut at an angle and metal bars were fitted, front view figure 1a) and side view figure 1b). A small piece of wire was wound around the metal bar to anchor the front incisors of the mouse. A piece of ribbon was attached to the platform to tie around the mouse to keep it in place during the procedure. 
<img src="/files/ftp_upload/2702/2702fig2.jpg" alt="Figure 2"/> 
Figure 2. Gavage needle for i.t injection. A gavage needle (right) was bent at an angle of &tilde;135&deg;(left) for i.t injection. 

<img src="/files/ftp_upload/2702/2702fig3.jpg" alt="Figure 3"/> 
Figure 3. Percentage of all CD11c+ cells in the lungs that harbor i.t injected GFP expressing Bacillus anthracis (Sterne-p6GFP), 5hrs post challenge. AJ mice were challenge with 1x108 GFP- Bacillus anthracis spores and the lungs were analyzed at 5hrs post infection. The dot plots show percentage of CD11c+ lung cells that are positive for Bacillus anthracis.
<img src="/files/ftp_upload/2702/2702fig4.jpg" alt="Figure 4"/> 
Figure 4. Detection of fluorescent beads (yellow/green) in the lung draining lymph nodes 24 hours following i.t injection. AJ mice were given either PBS (left panel) or 5x109 Fluoresbrite YG Microspheres (0.5&mu;m) with 10&mu;g LPS (right panel) i.t and LDLNs were analyzed 24hpi. Representative dot plots show percentage of CD11chi cells in the LDLNs that harbor fluorescent beads plotted against an empty channel.

<ol>
	<li>Grayson, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controls+for+lung+dendritic+cell+maturation+and+migration+during+respiratory+viral+infection.">Controls for lung dendritic cell maturation and migration during respiratory viral infection.</a> J Immunol. 179, 1438-1438 (2007).</li><li>GeurtsvanKessel, C. H., Lambrecht, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Division+of+labor+between+dendritic+cell+subsets+of+the+lung.">Division of labor between dendritic cell subsets of the lung.</a> Mucosal Immunol. 1, 442-442 (2008).</li><li>Bar-Haim, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interrelationship+between+dendritic+cell+trafficking+and+Francisella+tularensis+dissemination+following+airway+infection.">Interrelationship between dendritic cell trafficking and Francisella tularensis dissemination following airway infection.</a> PLoS Pathog. 4, e1000211-e1000211 (2008).</li><li>Kim, T. S., Braciale, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Respiratory+dendritic+cell+subsets+differ+in+their+capacity+to+support+the+induction+of+virus-specific+cytotoxic+CD8++T+cell+responses.">Respiratory dendritic cell subsets differ in their capacity to support the induction of virus-specific cytotoxic CD8+ T cell responses.</a> PLoS One. 4, e4204-e4204 (2009).</li><li>Bakocevic, N., Worbs, T., Davalos-Misslitz, A., Forster, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell-dendritic+cell+interaction+dynamics+during+the+induction+of+respiratory+tolerance+and+immunity.">T cell-dendritic cell interaction dynamics during the induction of respiratory tolerance and immunity.</a> J Immunol. 184, 1317-1317 (2010).</li><li>Thomas, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+particle+size+on+the+pathology+and+efficacy+of+vaccination+in+a+murine+model+of+inhalational+anthrax.">Influence of particle size on the pathology and efficacy of vaccination in a murine model of inhalational anthrax.</a> J Med Microbiol. , (2010).</li><li>Kiyono, H., Fukuyama, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NALT-+versus+Peyer's-patch-mediated+mucosal+immunity.">NALT- versus Peyer's-patch-mediated mucosal immunity.</a> Nat Rev Immunol. 4, 699-699 (2004).</li><li>Parungo, C. 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No conflicts of interest declared.

We have utilized this protocol to study trafficking of Bacillus anthracis spores from the lungs to the LDLNs. For 
similar applications, the number of particles delivered to the lungs should be carefully chosen so that the injected material can be detected 
in the LDLNs by flow cytometry. We have also successfully used this method for adoptive transfer of cells and labeling of specific lung cell 
populations using fluorescently labeled antibodies. In addition, this method is routinely used to administer bleomyci...

<table border="1">
 <tbody>
 <tr>
 <th>Name of the reagent</th>
 <th>Company</th>
 <th>Catalogue number</th>
 <th>Comments (optional)</th>
 </tr>
 <tr>
 <td>70&mu;m Cell stariner</td>
 <td>BD</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Aniline Blue</td>
 <td>Fisher</td>
 <td>A967-25</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Animal Feeding needle</td>
 <td>Popper and Sons Inc</td>
 <td>7920</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Collagenase</td>
 <td>Sigma</td>
 <td>C2139</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Collagenase TypeIV</td>
 <td>Worthington</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Collagenase D</td>
 <td>Roche</td>
 <td>11088974103</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>DPBS</td>
 <td>Invitrogen</td>
 <td>14190</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Fluoresbrite YG Microspheres (0.5&mu;m)</td>
 <td>Polysciences, Inc</td>
 <td>17152</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>HBSS without Calcium chloride and magnesium chloride</td>
 <td>Invitrogen</td>
 <td>14170</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Ketamine HCl &nbsp;(100mg/ml)</td>
 <td>Hospira Inc</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Laryngoscope Blade</td>
 <td>PennCentury, Inc</td>
 <td>For Model LS-1</td>
 <td>Refer to <a href="http://www.penncentury.com">www.penncentury.com</a></td>
 </tr>
 <tr>
 <td>Lightweight Fiber Optic Laryngoscope</td>
 <td>WelchAllyn</td>
 <td>80814</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Red Fluorescent Beads (0.5&mu;m)</td>
 <td>Invitrogen</td>
 <td>F8812</td>
 <td>For i.p injection</td>
 </tr>
 <tr>
 <td>Xylazine (100mg/ml)</td>
 <td>Lloyd Laboratories</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

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.

Watch this Scientific Journal Video about Non-surgical Intratracheal Instillation of Mice with Analysis of Lungs and Lung Draining Lymph Nodes by Flow Cytometry at JoVE.com

Non-surgical Intratracheal Instillation of Mice with Analysis of Lungs and Lung Draining Lymph Nodes 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, ...

non-surgical-intratracheal-instillation-mice-with-analysis-lungs-lung

Research

JoVE Journal

Immunology and Infection

57.2K Views.  University of Colorado School of Medicine. Illustriamo non chirurgica consegna dei materiali di prova nei polmoni di topi anestetizzati attraverso la trachea. Questo metodo permette l'esposizione dei polmoni a patogeni batterici e virali, citochine, anticorpi, perline, prodotti chimici o coloranti. Approfondiremo la raccolta ed elaborazione dei polmoni e dei linfonodi polmonari drenante (LDLNs) per la citometria a flusso.

Video: Non-surgical Intratracheal Instillation of Mice with Analysis of Lungs and Lung Draining Lymph Nodes by Flow Cytometry

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of phenol soluble modulins (PSMs) and other toxins produced and secreted by Staphylococcus aureus on neutrophils. To study the effects of the PSMs on neutrophils we isolate fresh neutrophils using density gradient centrifugation. These neutrophils are loaded with a dye that fluoresces upon calcium mobilization. The activation of neutrophils by PSMs initiates a rapid and transient increase in the free intracellular calcium concentration. In a flow cytometry experiment this rapid mobilization can be measured by monitoring the fluorescence of a pre-loaded dye that reacts to the increased concentration of free Ca2+. Using this method we can determine the PSM concentration necessary to activate the neutrophil, and measure the effects of specific and general inhibitors of the neutrophil activation.	
	To investigate the expression of the PSMs in the intracellular space, we have constructed reporter fusions of the promoter of the PSM&alpha; operon to GFP. When these reporter strains of S. aureus are phagocytosed by neutrophils, the induction of expression can be observed using fluorescence microscopy.

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of PSMs and other toxins secreted by Staphylococcus aureus on neutrophils using flow cytometry and fluorescence microscopy.

We present methods to study the effect of phenol soluble modulins (PSMs)  and other toxins produced and secreted by Staphylococcus  aureus on neutrophils. ...

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable method of analysis by flow cytometry to study nanoparticle interaction with immune cells. Primary immune cells can be easily purified from human or mouse tissues by antibody-mediated magnetic isolation. In the first instance, the different cell populations running in a flow cytometer can be distinguished by the forward-scattered light (FSC), which is proportional to cell size, and the side-scattered light (SSC), related to cell internal complexity. Furthermore, fluorescently labeled antibodies against specific cell surface receptors permit the identification of several subpopulations within the same sample. Often, all these features vary when cells are boosted by external stimuli that change their physiological and morphological state. Here, 50 nm FITC-SiO2 nanoparticles are used as a model to identify the internalization of nanostructured materials in human blood immune cells. The cell fluorescence and side-scattered light increase after incubation with nanoparticles allowed us to define time and concentration dependence of nanoparticle-cell interaction. Moreover, such protocol can be extended to investigate Rhodamine-SiO2 nanoparticle interaction with primary microglia, the central nervous system resident immune cells, isolated from mutant mice that specifically express the Green Fluorescent Protein (GFP) in the monocyte/macrophage lineage. Finally, flow cytometry data related to nanoparticle internalization into the cells have been confirmed by confocal microscopy.

Analysis of nanoparticle interaction with defined subpopulations of immune cells by flow cytometry.

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph node (DLN). The mobilization of DCs to the DLN is complex and remains to be fully elucidated during infection. Herein described is the use of an innovative, simple assay that relies on the fluorochrome 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) to track the migration of DCs during footpad infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin (BCG) in C57BL/6 mice. This assay enables the characterization of skin DC sub-populations that actively relocate to the draining, popliteal LN in response to BCG. This protocol originates from a BCG model where migratory skin DCs were identified by flow cytometry. The assay is amiable to the study and identification of DCs or other cells that home to the popliteal LN after inoculation of microbes, their metabolites or other inflammatory stimuli in the footpad, and consequently to study factors that regulate the migration of these cells.

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with &lt;em&gt;Mycobacterium bovis&lt;/em&gt; Bacille Calmette-Gu&amp;#233;rin

An assay in mice to track cell migration from skin to draining lymph node is described which enables the characterization of skin Dendritic cells mobilized to the lymph node after footpad infection with Bacille Calmette-Gu&#233;rin.

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

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 ...

Collection and Processing of Lymph Nodes from Large Animals for RNA Analysis: Preparing for Lymph Node Transcriptomic Studies of Large Animal Species

Large animals (both livestock and wildlife) serve as important reservoirs of zoonotic pathogens, including Brucella, Mycobacterium bovis, Salmonella, and E. coli, and are useful for the study of pathogenesis and/or spread of the bacteria in natural hosts. With the key function of lymph nodes in the host immune response, lymph node tissues serve as a potential source of RNA for downstream transcriptomic analyses, in order to assess the temporal changes in gene expression in cells over the course of an infection. This article presents an overview of the process of lymph node collection, tissue sampling, and downstream RNA processing in livestock, using cattle (Bos taurus) as a model, with additional examples provided from the American bison (Bison bison). The protocol includes information about the location, identification, and removal of lymph nodes from multiple key sites in the body. Additionally, a biopsy sampling methodology is presented that allows for a consistency of sampling across multiple animals. Several considerations for sample preservation are discussed, including the generation of RNA suitable for downstream methodologies like RNA-sequencing and RT-PCR. Due to the long delays inherent in large animal vs. mouse time course studies, representative results from bison and bovine lymph node tissues are presented to describe the time course of the degradation in this tissue type, in the context of a review of previous methodological work on RNA degradation in other tissues. Overall, this protocol will be useful to both veterinary researchers beginning transcriptome projects on large animal samples and to molecular biologists interested in learning techniques for in vivo tissue sampling and in vitro processing.

This protocol provides an overview of procedures for the isolation of RNA for the transcriptomic profiling of lymph node tissues from large animals, including steps in the identification and excision of lymph nodes from livestock and wildlife, sampling approaches to provide consistency across multiple animals, and considerations plus representative results for the post-collection preservation and processing for RNA analysis.

Large animals (both livestock and wildlife) serve as important reservoirs of zoonotic pathogens, including Brucella, Mycobacterium bovis, Salmonella, and ...

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 Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can have pathological significance. An ideal method for examining alterations to monocytes is whole blood flow cytometry as the minimal handling of samples by this method limits artifactual cell activation. However, many different approaches are taken to gate the monocyte subsets leading to inconsistent identification of the subsets between studies. Here we demonstrate a method using whole blood flow cytometry to identify and characterize human monocyte subsets (classical, intermediate, and non-classical). We outline how to prepare the blood samples for flow cytometry, gate the subsets (ensure contaminating cells have been removed), and determine monocyte subset expression of surface markers &#8212; in this example M1 and M2 markers. This protocol can be extended to other studies that require a standard gating method for assessing monocyte subset proportions and monocyte subset expression of other functional markers.

Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory).

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...

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, ...

Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a myeloid-biased 39-antibody CyTOF panel to evaluate the hematopoietic system with a focus on the neutrophil-lineage cells by using this protocol. The CyTOF result was analyzed with an open-resource dimensional reduction algorithm, viSNE, and the data was presented to demonstrate the outcome of this protocol. We have discovered new neutrophil-lineage cell populations based on this protocol. This protocol of fresh whole BM preparation may be used for 1), CyTOF analysis to discover unidentified cell populations from whole BM, 2), investigating whole BM defects for patients with blood disorders such as leukemia, 3), assisting optimization of fluorescence-activated flow cytometry protocols that utilize fresh whole BM.

Here, we present a protocol to process fresh bone marrow (BM) isolated from mouse or human for high-dimensional mass cytometry (Cytometry by Time-Of-Flight, CyTOF) analysis of neutrophil-lineage cells.

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a ...

A Model of Self-limited Acute Lung Injury by Unilateral Intra-bronchial Acid Instillation

Selective intra-bronchial instillation of hydrochloric acid (HCl) to the murine left mainstem bronchus causes acute tissue injury with histopathologic findings similar to human acute respiratory distress syndrome (ARDS). The resulting alveolar edema, alveolar-capillary barrier damage, and leukocyte infiltration predominantly affect the left lung, preserving the right lung as an uninjured control and allowing animals to survive. This model of self-limited acute lung injury enables investigation of tissue resolution mechanisms, such as macrophage efferocytosis of apoptotic neutrophils and restitution of alveolar-capillary barrier integrity. This model has helped identify important roles for resolution agonists, including specialized pro-resolving mediators (SPMs), providing a foundation for the development of new therapeutic approaches for patients with ARDS.

Selective intra-bronchial acid instillation to the left lung in mice results in unilateral and self-limited acute lung injury that models human acute respiratory distress syndrome (ARDS) induced by gastric acid aspiration.

Selective intra-bronchial instillation of hydrochloric acid (HCl) to the murine left mainstem bronchus causes acute tissue injury with histopathologic ...