R.Z. was supported by the National Natural Science Foundation of China (No. 81871258) and funds provided by Fourth Military Medical University (No.2020rcfczr). Y.Z. was supported by the Natural Science Basic Research Program of Shaanxi (No. 2021JM-081).

All experiments were performed in accordance with the Care and Use of Laboratory Animals Guidelines. All procedures and protocols were approved by the Scientific Research Ethics Committee of the Fourth Military Medical University (No. 20211008).
1. Murine AR model establishment
<ol>
	<li>House male and female wild-type (WT) C57BL/6 mice aged 8-12 weeks under specific pathogen-free conditions and provide standard laboratory chow and water.</li>
	<li>Emulsify 50 &#181;g of ovalbumin (OVA) in 0.2 mL of sterile PBS containing 2 mg of aluminum hydroxide on a clean bench to maintain sterility. On days 0, 7, and 14, intraperitoneally inject female or male mice with 50 &#181;g each of emulsified OVA.</li>
	<li>On days 21, 22, 23, 24, and 25, intranasally instill mice with 50 &#181;g of OVA dissolved in 30 &#181;L of sterile PBS (15&#160;&#181;L per nostril) under inhalation anesthesia (2-3% isoflurane with an Oxygen flow rate or 0.5 L/min).</li>
	<li>Euthanize&#160;the mice 24 h after the last challenge (day 26).</li>
</ol>
2. Isolation of mononuclear cells (MNCs) from the nasal mucosa
<ol>
	<li>Euthanize the mice by cervical dislocation under deep anesthesia. Soak the mice head up in 75% ethanol for 5 min and avoid ethanol entering the external nostril. Place the abdomen downward on the operating table.</li>
	<li>Cut off the fore-teeth. At the midline of the head, make an incision, and cut open the skin using scissors.</li>
	<li>Remove the lower jaw, cut off the entire nose along the end of the palate, and place the tissue into a 60 mm Petri dish containing 5 mL of ice-cold PBS. Using scissors and forceps, remove the flesh and muscles adhering to the bones.</li>
	<li>Transfer the mouse nose to a new 60 mm Petri dish containing 5 mL of ice-cold PBS. Wash the bones twice with 5 mL of ice-cold PBS.</li>
	<li>Transfer the nose into a 1.5 mL microcentrifuge tube.</li>
	<li>Sufficiently smash and transfer the nose to a 15 mL tube containing 2 mL of prewarmed digest buffer (RPMI 1640 medium supplemented with 1 mg/mL of collagenase IV and 10 U/mL DNase I).</li>
	<li>Fasten the lid and place the tube vertically in an orbital shaker at 37 &#176;C, with continuous agitation at 120-150 rpm for 40 min. 
	NOTE: Pre-warm the digest buffer to 37 &#176;C to achieve the highest enzyme activity.</li>
	<li>Add 5 mL of ice-cold RPMI 1640 medium containing 10% fetal bovine serum (FBS) to stop the digestion process.</li>
	<li>Filter through a 70 &#181;m cell strainer to remove solid fragments.</li>
	<li>Centrifuge at 500 x g for 5 min, and then gently discard the supernatant.</li>
	<li>Resuspend the pellet in ice-cold RPMI 1640 medium and centrifuge at 500 x g for 5 min. Gently discard the supernatant.</li>
	<li>Resuspend the cell pellet in 4 mL of 40% density gradient media (160 &#181;L of 10x PBS + 1.44 mL of density gradient media stock solution + 2.4 mL of RPMI 1640 medium).</li>
	<li>Gently insert a Pasteur pipette to the bottom of the tube and slowly add 2.5 mL of 80% density gradient media (200 &#181;L of 10x PBS + 1.8 mL of density gradient media stock solution + 0.5 mL of RPMI 1640 medium).</li>
	<li>Set the acceleration and deceleration rate of the centrifuge lower than the third gear and centrifuge at 400 x g for 15 min at room temperature (RT).</li>
	<li>Remove the top layer of impurities before draining the cells at the interface to avoid potential contamination. Carefully drain the mononuclear cells (MNCs) layer at the 40%/80% density gradient media interface into a 15 mL tube containing 2 mL of ice-cold PBS using a pipette. Wash the cells with ice-cold PBS twice.</li>
</ol>
3. Surface staining for FCM analysis
<ol>
	<li>Harvest the cells and centrifuge at 500 x g for 5 min at 4 &#176;C. Resuspend the cell pellet in staining buffer (PBS supplemented with 2% FBS and 0.1% NaN3) and centrifuge at 500 x g for 5 min at 4 &#176;C. Discard the supernatant. 
	CAUTION: Be cautious when preparing the staining buffer. NaN3 is very toxic and may cause damage to organs if swallowed, inhaled, or in contact with skin. Also, avoid release to the environment.</li>
	<li>Resuspend the cells in 80 &#181;L blocking solution (anti-CD16/32 antibody (1:100) diluted in staining buffer) per tube. Incubate for 30 min in the dark at 4 &#176;C.</li>
	<li>Without washing, add 20 &#181;L of the appropriate dilution of the surface-staining antibody cocktail (Table 1).</li>
	<li>Add 1 &#181;L (per test) of fixable viability dye 520 (FVD) just before adding the antibody cocktail to the samples. Incubate in the dark at 4 &#176;C for 30 min. Set matching isotype antibodies and fluorescence minus one (FMO) as the negative controls.</li>
	<li>Wash the cells in 500 &#181;L of staining buffer by centrifuging at 500 x g for 5 min at 4 &#176;C.</li>
	<li>Resuspend the cell pellet in 200 &#181;L of staining buffer. Add 50 &#181;L of vortexed absolute counting beads to the stained cells and agitate. Subject them to a flow cytometry (FCM) analysis. 
	NOTE: FCM data were obtained using a spectral cell analyzer and analyzed with flow cytometry (FCM) analysis software.</li>
</ol>

The authors have nothing to disclose.

ILC2s are closely associated with type 2 inflammation and inflammatory disorders, as demonstrated by an increasing number of studies. Both mouse models and human observation contribute to a better understanding of its function in the upper airway. In asthma pathophysiology, ILC2s are activated through thymic stromal lymphopoietin, IL-25, and IL-33, which are mostly produced by epithelial cells. Then mirroring Th2 cells, ILC2s produce IL-4, IL-5, and IL-13 to aggravate type 2 inflammation32. Furthe...

Group 2 innate lymphoid cells (ILC2s) were first discovered in the peritoneal cavity tissues of mice and were subsequently demonstrated to be present in the blood and other peripheral tissues such as the lungs, skin, and nasal cavity1,2,3. As tissue-resident cells, ILC2s are mainly maintained and proliferated locally and function as the first guards responding to exogenous harmful stimuli through producing numerous type 2 cytokines and inducing type 2 immunity4,5,6. ILC2s can also exert their effects by trafficking toward the infected tissues7,8.
Similar to T-helper 2 (Th2) cells, the complicated regulatory networks of ILC2s ensure their significant involvement in the progression of various type 2 inflammatory diseases, including airway allergic diseases8,9. In asthma, epithelial cell-derived alarmins can activate ILC2s, which further promote pulmonary inflammation through the secretion of interleukin (IL)-4, IL-5, and IL-1310. Clinical studies have also indicated that ILC2 levels were significantly elevated in the sputum and blood of patients with severe asthma, suggesting an association of ILC2s with asthma severity and their function as a predictor of asthma progression11.
Allergic rhinitis (AR) is a common chronic inflammatory disease that affects millions of people annually, and effective treatments for this disease are limited12,13. ILC2s play crucial roles in the pathophysiology of AR, whether in the sensitization phase or symptom generation and inflammation phase14. In patients with AR, the levels of ILC2 in the peripheral blood have been reported to be elevated both locally and systemically15. However, certain effects and the underlying mechanisms of ILC2s on the pathophysiology and progression of AR still require further exploration.
CD226 - a transmembrane glycoprotein that serves as a costimulatory molecule - is primarily expressed on natural killer (NK) cells, T cells, and other inflammatory monocytes16,17. The interaction of CD226 and its ligands (CD155 and/or CD112) or competitor (TIGIT) allows it to participate in the biological functions of various immune cells18. The binding of the ligands on antigen-presenting cells to CD226 on cytotoxic lymphocyte (CTL) promotes the activation of both cells simultaneously, while the activation of CTL can be further suppressed by TIGIT (T cell immunoreceptor with Ig and ITIM domains), the competitor of CD22619,20. A human ex vivo study revealed that CD226 and CD155 on T cells regulate the balance between Th1/Th17 and Th2 through differentially modulating Th subsets21. CD226 can likewise mediate platelet adhesion and NK tumor-killing activity22,23. Meanwhile, CD226 is well-studied in the pathogenesis of various infectious diseases, autoimmune diseases, and tumors18,24,25. At present, CD226 has become a new bright spot for immunotherapy. Studies have found that extracellular vesicles can reverse CD226 expression on NK cells to reinstate their cytotoxic activity and intervene in the progression of lung cancer26. A recent study has revealed a subcluster of fetal intestinal group 3 ILCs characterized with high CD226 expression by single-cell RNA sequencing27, which indicated that CD226 might exert roles in the innate lymphoid cell-mediated immunity.
Our knowledge about ILC2s in airway inflammation is primarily based on studies on asthma; however, little is known about their functions in nasal mucosal immunity. Thus, a protocol was established to isolate and identify ILC2s from the nasal mucosa. The study focuses on the expression of CD226 on ILC2s in nasal tissues and its variation between healthy and AR mice. This may provide novel insights into the underlying mechanisms of ILC2-mediated regulation in the local immunity and serve as a basis for developing new approaches for AR treatment.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Aluminum hydroxide</td>
			<td>Meilun biological Technology</td>
			<td>21645-51-2</td>
			<td></td>
		</tr>
		<tr>
			<td>CD11b</td>
			<td>eBioscience</td>
			<td>11-0112-82</td>
			<td>Used in antibody coctail</td>
		</tr>
		<tr>
			<td>CD11c</td>
			<td>BioLegend</td>
			<td>117306</td>
			<td>Used in antibody coctail</td>
		</tr>
		<tr>
			<td>CD16/32</td>
			<td>BioLegend</td>
			<td>101302</td>
			<td>Clone: 93; Dilution 1:100</td>
		</tr>
		<tr>
			<td>CD226</td>
			<td>BioLegend</td>
			<td>128812</td>
			<td>Used in antibody coctail</td>
		</tr>
		<tr>
			<td>CD3e</td>
			<td>BioLegend</td>
			<td>100306</td>
			<td>Used in antibody coctail</td>
		</tr>
		<tr>
			<td>CD45</td>
			<td>BioLegend</td>
			<td>103128</td>
			<td>Used in antibody coctail</td>
		</tr>
		<tr>
			<td>CD45R</td>
			<td>eBioscience</td>
			<td>11-0452-82</td>
			<td>Used in antibody coctail</td>
		</tr>
		<tr>
			<td>CD90.2</td>
			<td>BD Pharmingen</td>
			<td>553014</td>
			<td>Used in antibody coctail</td>
		</tr>
		<tr>
			<td>Collagenase IV</td>
			<td>DIYIBio</td>
			<td>DY40128</td>
			<td></td>
		</tr>
		<tr>
			<td>CountBright absolute counting beads</td>
			<td>Invitrogen</td>
			<td>C36950</td>
			<td>absolute counting beads</td>
		</tr>
		<tr>
			<td>Dnase <img alt="Equation 1" src="/files/ftp_upload/63525/63525eq01v2.jpg" style="margin: auto;" /></td>
			<td>Beyotime</td>
			<td>D7076</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>gibco</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr>
			<td>Fixable Viability Dye eFluor 520 (FITC)</td>
			<td>eBioscience</td>
			<td>65-0867-14</td>
			<td>FVD</td>
		</tr>
		<tr>
			<td>HBSS, calcium, magnesium</td>
			<td>Servicebio</td>
			<td>G4204-500</td>
			<td></td>
		</tr>
		<tr>
			<td>KLRG1</td>
			<td>eBioscience</td>
			<td>17-5893-81</td>
			<td>Used in antibody coctail</td>
		</tr>
		<tr>
			<td>NaN3</td>
			<td>SIGMA</td>
			<td>S2002</td>
			<td></td>
		</tr>
		<tr>
			<td>NovoExpress software</td>
			<td>AgilentTechnologies</td>
			<td>Version 1.5.0</td>
			<td>flow cytometry (FCM) analysis software</td>
		</tr>
		<tr>
			<td>OVA</td>
			<td>SIGMA</td>
			<td>9006-59-1</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS, 1x</td>
			<td>Servicebio</td>
			<td>G4202-500</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS, 10x</td>
			<td>Servicebio</td>
			<td>G4207-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll</td>
			<td>Yeasen</td>
			<td>40501ES60</td>
			<td>density gradient media</td>
		</tr>
		<tr>
			<td>RPMI 1640 culture media</td>
			<td>Corning</td>
			<td>10-040-CVRV</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectral cell analyzer</td>
			<td>SONY</td>
			<td>SA3800</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of group 2 innate lymphoid cells from mouse nasal mucosa to detect the expression of cd226

With abundant research on group 2 innate lymphoid cells (ILC2s) published over the years, ILC2s are widely known to be implicated in regulating various pathological processes, including anti-helminth immunity, tissue repair, thermogenesis, and autoimmune diseases such as asthma and allergic rhinitis (AR). ILC2s permanently reside in peripheral tissues such as the skin, gut, lungs, and nasal cavity; however, there is limited information about their exact functions in nasal mucosal immunity. CD226 is an activating costimulatory molecule, mainly expressed on natural killer (NK) cells, T cells, and inflammatory monocytes. However, whether ILC2s express CD226 or play a role in the pathogenesis of ILC2s-related diseases remains unknown. Here, we established a method to isolate and identify ILC2s from the nasal mucosa and detected CD226 expression on ILC2s obtained from healthy and AR mice. Herein, we describe this protocol for the isolation and identification of ILC2s from mouse nasal mucosa, which will help explore the internal pathological mechanism of immunological disorders in nasal mucosal diseases.

An OVA-induced murine model was developed to explore the role of ILC2s in AR. The construction of AR murine model was based on previous studies with slight modifications28,29,30,31. A 10 min video was captured to measure the frequency of sneezing and nasal scratching after the last nasal challenge. Allergic symptoms of the OVA-induced-AR mice were presented in Figure 1</str...

Watch this Scientific Journal Video about Isolation of Group 2 Innate Lymphoid Cells from Mouse Nasal Mucosa to Detect the Expression of CD226 at JoVE.com

Isolation of Group 2 Innate Lymphoid Cells from Mouse Nasal Mucosa to Detect the Expression of CD226

Group 2 innate lymphoid cells (ILC2s), implicated in type 2 inflammation, mainly participate in response to helminth infection, allergic diseases, metabolic homeostasis, and tissue repair. In this study, a procedure to isolate ILC2s from murine nasal mucosa and detect the expression of CD226 is demonstrated.

With abundant research on group 2 innate lymphoid cells (ILC2s) published over the years, ILC2s are widely known to be implicated in regulating various ...

isolation-group-2-innate-lymphoid-cells-from-mouse-nasal-mucosa-to

Research

JoVE Journal

Immunology and Infection

1.7K Views.  Fourth Military Medical University. Group 2 innate lymphoid cells (ILC2s), implicated in type 2 inflammation, mainly participate in response to helminth infection, allergic diseases, metabolic homeostasis, and tissue repair. In this study, a procedure to isolate ILC2s from murine nasal mucosa and detect the expression of CD226 is demonstrated.

Video: Isolation of Group 2 Innate Lymphoid Cells from Mouse Nasal Mucosa to Detect the Expression of CD226

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and food antigens while concomitantly remaining poised to mount inflammatory responses toward invasive pathogens1,2. Antigen presenting cells, particularly DCs and macrophages, play critical roles in maintaining intestinal immune homeostasis via their ability to sense and appropriately respond to the microbiota3-14. Efficient isolation of intestinal DCs and macrophages is a critical step in characterizing the phenotype and function of these cells. While many effective methods of isolating intestinal immune cells, including DCs and macrophages, have been described6,10,15-24, many rely upon long digestions times that may negatively influence cell surface antigen expression, cell viability, and/or cell yield. Here, we detail a methodology for the rapid isolation of large numbers of viable, intestinal DCs and macrophages. Phenotypic characterization of intestinal DCs and macrophages is carried out by directly staining isolated intestinal cells with specific fluorescence-labeled monoclonal antibodies for multi-color flow cytometric analysis. Furthermore, highly pure DC and macrophage populations are isolated for functional studies utilizing CD11c and CD11b magnetic-activated cell sorting beads followed by cell sorting.

Here, we detail a methodology for the rapid isolation of mouse intestinal dendritic cells (DCs) and macrophages. Phenotypic characterization of intestinal DCs and macrophages is performed using multi-color flow cytometric analysis while magnetic bead enrichment followed by cell sorting is used to yield highly pure populations for functional studies.

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.

Bone marrow transplantation provides a way to change the genotype of the bone marrow derived cells. If the gene of interest is expressed in both bone marrow derived cells and non-bone marrow derived cells, bone marrow transplantation can change the bone marrow derived cells to a different genotype without changing the non-bone marrow derived cell genotype.

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice ...

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and bacteria 1. Mucosae are also inductive sites in the host to generate immunity against pathogens, such as the Peyers patches in the intestinal tract and the nasal-associated lymphoreticular tissue in the respiratory tract. This unique feature brings mucosal immunity as a crucial player of the host defense system. Many studies have been focused on gastrointestinal and respiratory mucosal sites. However, there has been little investigation of reproductive mucosal sites. The genital tract mucosa is the primary infection site for sexually transmitted diseases (STD), including bacterial and viral infections. STDs are one of the most critical health challenges facing the world today. Centers for Disease Control and Prevention estimates that there are 19 million new infectious every year in the United States. STDs cost the U.S. health care system $17 billion every year 2, and cost individuals even more in immediate and life-long health consequences. In order to confront this challenge, a greater understanding of reproductive mucosal immunity is needed and isolating lymphocytes is an essential component of these studies. Here, we present a method to reproducibly isolate lymphocytes from murine female genital tracts for immunological studies that can be modified for adaption to other species. The method described below is based on one mouse.&#x2003;

An efficient way to isolate lymphocytes from mouse genital tract is described. This method takes advantage of enzyme digestion and Percoll gradient separation to allow efficient isolation. This technique is also adaptable to for use in other species

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and new strains are continually emerging. Swine are able to be infected by diverse lineages of influenza A virus making them important hosts for the emergence and maintenance of novel influenza A virus strains. Sampling pigs in diverse settings such as commercial swine farms, agricultural fairs, and live animal markets is important to provide a comprehensive view of currently circulating IAV strains. The current gold-standard ante-mortem sampling technique (i.e. collection of nasal swabs) is labor intensive because it requires physical restraint of the pigs. Nasal wipes involve rubbing a piece of fabric across the snout of the pig with minimal to no restraint of the animal. The nasal wipe procedure is simple to perform and does not require personnel with professional veterinary or animal handling training. While slightly less sensitive than nasal swabs, virus detection and isolation rates are adequate to make nasal wipes a viable alternative for sampling individual pigs when low stress sampling methods are required. The proceeding protocol outlines the steps needed to collect a viable nasal wipe from an individual pig.

The authors present a protocol to collect swine nasal wipes to detect and isolate influenza A viruses.

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Mouse Models Of Helicobacter Infection And Gastric Pathologies

Helicobacter pylori is a gastric pathogen that is present in half of the global population and is a significant cause of morbidity and mortality in humans. Several mouse models of gastric Helicobacter infection have been developed to study the molecular and cellular mechanisms whereby H. pylori bacteria colonize the stomach of human hosts and cause disease. Herein, we describe protocols to: 1) prepare bacterial suspensions for the in vivo infection of mice via intragastric gavage; 2) determine bacterial colonization levels in mouse gastric tissues, by polymerase chain reaction (PCR) and viable counting; and 3) assess pathological changes, by histology. To establish Helicobacter infection in mice, specific pathogen-free (SPF) animals are first inoculated with suspensions (containing &#8805;105 colony-forming units, CFUs) of mouse-colonizing strains of either Helicobacter pylori or other gastric Helicobacter spp. from animals, such as Helicobacter felis. At the appropriate time-points post-infection, stomachs are excised and dissected sagittally into two equal tissue fragments, each comprising the antrum and body regions. One of these fragments is then used for either viable counting or DNA extraction, while the other is subjected to histological processing. Bacterial colonization and histopathological changes in the stomach may be assessed routinely in gastric tissue sections stained with Warthin-Starry, Giemsa or Haematoxylin and Eosin (H&#38;E) stains, as appropriate. Additional immunological analyses may also be undertaken by immunohistochemistry or immunofluorescence on mouse gastric tissue sections. The protocols described below are specifically designed to enable the assessment in mice of gastric pathologies resembling those in human-related H. pylori diseases, including inflammation, gland atrophy and lymphoid follicle formation. The inoculum preparation and intragastric gavage protocols may also be adapted to study the pathogenesis of other enteric human pathogens that colonize mice, such as Salmonella Typhimurium or Citrobacter rodentium.

Mouse Models Of Helicobacter Infection And Gastric Pathologies

Mice represent an invaluable in vivo model to study infection and diseases caused by gastrointestinal microorganisms. Here, we describe the methods used to study bacterial colonization and histopathological changes in mouse models of Helicobacter pylori-related disease.

Helicobacter pylori is a gastric pathogen that is present in half of the global population and is a significant cause of morbidity and mortality in ...

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Histocultures allow studying intercellular interactions within human tissues, and they can be employed to model host-pathogen interactions under controlled laboratory conditions. Ex vivo infection of human tissues with human immunodeficiency virus (HIV), among other viruses, has been successfully used to investigate early disease pathogenesis, as well as a platform to test the efficacy and toxicity of antiviral drugs. In the present protocol, we explain how to process and infect with HIV-1 tissue explants from human tonsils and cervical mucosae, and maintain them in culture on top of gelatin sponges at the liquid-air interface for about two weeks. This non-polarized culture setting maximizes access to nutrients in culture medium and oxygen, although progressive loss of tissue integrity and functional architectures remains its main limitation. This method allows monitoring HIV-1 replication and pathogenesis using several techniques, including immunoassays, qPCR, and flow cytometry. Of importance, the physiologic variability between tissue donors, as well as between explants from different areas of the same specimen, may significantly affect experimental results. To ensure result reproducibility, it is critical to use an adequate number of explants, technical replicates, and donor-matched control conditions to normalize the results of the experimental treatments when compiling data from multiple experiments (i.e., conducted using tissue from different donors) for statistical analysis.

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Infection of human tissues with human immunodeficiency virus (HIV) ex vivo provides a valuable 3D model of virus pathogenesis. Here, we describe a protocol to process and infect tissue specimens from human tonsils and female genital mucosae with HIV-1 and maintain them in culture at the liquid-air interface.

Histocultures allow studying intercellular interactions within human tissues, and they can be employed to model host-pathogen interactions under ...

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of glass-supported planar bilayers and in vitro-derived T-cell clones or lines1,2,3,4. How these findings apply to the primary human T cells isolated from blood or lymphoid tissues is not known, partly due to significant difficulties in obtaining a sufficient number of cells for analysis5. Here we address this through the development of a technique exploiting multichannel flow slides to build planar lipid bilayers containing activating and adhesion molecules. The low height of the flow slides promotes rapid cell sedimentation in order to synchronize cell:bilayer attachment, thereby allowing researchers to study the dynamic of the synaptic interface formation and the kinetics of the granules release. We apply this approach to analyze the synaptic interface of as few as 104 to 105 primary cryopreserved T cells isolated from lymph nodes (LN) and peripheral blood (PB). The results reveal that the novel planar lipid bilayer technique enables the study of the biophysical properties of primary human T cells derived from blood and tissues in the context of health and disease.

The protocol describes a technique to study the ability of primary polyclonal human T cells to form synaptic interfaces using planar lipid bilayers. We use this technique to show the differential synapse formation capability of human primary T cells derived from lymph nodes and peripheral blood.

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of ...

Isolation and Quantitative Evaluation of Brush Cells from Mouse Tracheas

Tracheal brush cells are cholinergic chemosensory epithelial cells poised to transmit signals from the airway lumen to the immune and nervous systems. They are part of a family of chemosensory epithelial cells which include tuft cells in the intestinal mucosa, brush cells in the trachea, and solitary chemosensory and microvillous cells in the nasal mucosa. Chemosensory cells in different epithelial compartments share key intracellular markers and a core transcriptional signature, but also display significant transcriptional heterogeneity, likely reflective of the local tissue environment. Isolation of tracheal brush cells from single cell suspensions is required to define the function of these rare epithelial cells in detail, but their isolation is challenging, potentially due to the close interaction between tracheal brush cells and nerve endings or due to airway-specific composition of tight and adherens junctions. Here, we describe a procedure for isolation of brush cells from mouse tracheal epithelium. The method is based on an initial separation of tracheal epithelium from the submucosa, allowing for a subsequent shorter incubation of the epithelial sheet with papain. This procedure offers a rapid and convenient solution for flow cytometric sorting and functional analysis of viable tracheal brush cells.

Brush cells are rare cholinergic chemosensory epithelial cells found in the na&#239;ve mouse trachea. Due to their limited numbers, ex vivo evaluation of their functional role in airway immunity and remodeling is challenging. We describe a method for isolation of tracheal brush cells by flow cytometry.

Tracheal brush cells are cholinergic chemosensory epithelial cells poised to transmit signals from the airway lumen to the immune and nervous systems. ...