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. Furthermore, different subsets of ILC2s also produce IL-10 and IL-1733,34,35. AR is also a type of airway allergic inflammation driven by type 2 cells. The participation of ILC2s in AR pathophysiology has been demonstrated recently14. However, the potential regulatory mechanisms of ILC2s under AR remain largely unknown.
Evidence indicates that the interaction between different ILC2s may rely on the surface molecules36. For instance, both ICOS and ICOS-ligands expressed on ILC2s are necessary for their survival and functions37. In addition, ILC2s also express ICAM-1 and LFA-1, which exert important effects on cell migration and cell-cell interaction7,38. In addition, ILC2s can crosstalk with other immune cells through these cell-surface molecules. For instance, OX40L is a dominant mediator for the interaction between CD4+ T cells and ILC2s in lung inflammation39. Because CD226, as a transmembrane costimulatory molecule, expresses on various immune cells, we also evaluated its expression on ILC2s in nasal tissues from healthy and AR mice in this study.
General OVA-induced AR modeling includes intraperitoneally systemic sensitization and intranasally local stimulation, which usually require at least 3-4 weeks to achieve satisfactory modeling results31. Another OVA-induced nasal-only delivery AR model takes a similar time as the general model method to achieve an approximate level of type 2 inflammation and AR symptoms40. However, the number and function of nasal ILC2s by these two AR modeling methods still need further research. We assessed sneezing and nasal rubbing to evaluate AR symptoms. Although it would be more convincing to include the assessment for rhinorrhea, we found it is hard to catch sight of the rhinorrhea without interfering with mice behavior during evaluation, so we regrettably excluded these criteria. In addition, the evaluation for sneezing and nasal rubbing might be profound enough to illustrate the severity of murine AR symptoms for many of the relevant articles that employed only these two criteria41,42,43.
Previous studies about mice nasal ILC2s have described the isolation of murine nasal mucosa44,45. Their protocol is as follows: Briefly, remove the soft tissue around the skull, open the nasal bone to expose the nasal cavity, and scrape the nasal mucosal tissue off the surface of the skull with forceps. While most of the isolated mucosa was used for IF, IHC, or qPCR, little is reported about the following isolation of mononuclear cells (MNCs) and FCM analysis. In this study, considering the low amounts of ILC2s located in the nasal mucosa, the operation retained the maximum volume of the nasal mucosa by digesting the entire nose instead of stripping the mucosa from the surface of the nasal bone. To optimize the digestion protocol, we evaluated three digest media: 0.5 mg/mL of collagenase IV, 1 mg/mL of collagenase IV, and 1 mg/mL of collagenase IV and 10 U/mL DNase I, and compared the percentage of living cells and lymphocyte frequency obtained by these three digest media. The proportion of living cells acquired by all three media has no difference (90%-95%); however, the first two types of digestion were inadequate with lower lymphocyte frequency (~5%). Thus, 1 mg/mL of collagenase IV and 10 U/mL of DNase I were chosen as the digest media in this study. However, other enzymes such as liberase and dispase that may replace collagenase have not been studied. Since the type and concentration of enzymes are critical for cell viability and cell-surface molecule expression46,47, the usage and dosage of these alternatives during nose digestion still need careful exploration and optimization. We used FVD eFluor 520 to check cell viability, because it can be detected using a 530/30 band pass filter which is equivalent to FITC, so we could exclude the dead cells while gating Lin- cells. Other cell viability dyes are also optional according to specific antibody panels. Furthermore, instead of intracellular staining of transcription factors GATA3, which is usually performed in other ILC2-related studies, ILC2s in the present study were characterized based on cell-surface markers such as Lin (CD11b, CD11c, CD3, and B220), CD45, CD90.2, and KLRG1 in FCM analysis. Among them, KLRG1 expressed on ILC2s has been identified as a specific marker of mature tissue ILC2s48,49. Although CD90.2 is less specific, it is still accepted as an ILC2-associated surface marker50. In this study, mice were not distinguished by sex. Research has shown that the abundance and cell-surface antigens of pulmonary ILCs in mice are different in gender, but the correlation of nasal ILC2s with gender still needs to be studied50. In addition, the protocol did not focus on the proportion of other hematopoietic cells. Different FCM antibody panels would help to solve this issue.
This protocol described a method to isolate ILC2s from the murine nasal mucosa. The isolated ILC2s can be stained for FCM characterization, sorted for cell culture, or stimulated in vitro for further analyses. Consistent with the findings of the previous studies10,45,51, a substantial accumulation of ILC2s was observed in the nasal mucosa of AR mice. Notably, CD226 was expressed on local ILC2s, and its level significantly increased further in allergic conditions. Therefore, the findings in this study suggest the involvement of CD226 in ILC2-dependent AR. Whether ILC2s in patients with AR express CD226 and whether the CD226 expression on ILC2s increases in allergic conditions should be investigated in future studies.
In conclusion, we established a protocol to isolate and identify ILC2s from the nasal mucosa and detected the expression of adhesion molecules within them. The protocol requires further optimization; however, the lack of studies that systematically and minutely describe the procedure for isolating and characterizing lymphocytes, particularly innate lymphoid cells in the nasal mucosa, makes this protocol a preliminary reference for researchers exploring the local nasal immunological environment.

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><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 &lt;img alt=&quot;Equation 1&quot; src=&quot;/files/ftp_upload/63525/63525eq01v2.jpg&quot; style=&quot;margin: auto;&quot;/&gt;</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>NaN&lt;sub&gt;3&lt;/sub&gt;</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. The frequency of nasal rubbing and sneezing in the AR mice was significantly higher than in the control group.
Next, we described a procedure to isolate MNCs from murine nasal mucosa and perform their characterization using FCM analysis. About 2-3 x 106 lymphocytes were obtained from each murine nose. The dead cells were excluded by FVD staining. The gating strategy for ILC2s among the isolated cells was Lin (CD11b, CD11c, CD3, and B220)&#8722; CD45+ CD90.2+ KLRG1+ cells. All gates were drawn based on isotype or FMO control. Typically, ~2%-3% of Lin&#8722; CD45+ cells were identified in the nasal mucosa of healthy control mice (Figure 2). The absolute number of ILC2s in murine nasal mucosa varies from 2,000-4,000.
We then isolated the ILC2s from the AR mice using the above-described method. There was no difference in the absolute numbers of lymphocytes between HC and AR mice. The FCM results indicated that the fraction of ILC2s in the AR mice was 9%-14% of the total number of Lin&#8722; CD45+ lymphocytes, indicating a remarkable increase in the number of ILC2s in AR nasal mucosa compared with that in healthy control mice (Figure 3).
We detected the expression of CD226 on the ILC2s. On average, 51.9% of ILC2s expressed CD226 under unimmunized conditions, whereas the average ratio of ILC2s expressing CD226 was 54.8% in the AR mice, which showed a nonsignificant tendency to increase compared with that in control mice. In addition to cell frequency, the cell-surface expression level of CD226 was also determined using mean fluorescence intensity (MFI) that was auto-calculated by FCM analysis software. Interestingly, the MFI of CD226 was significantly upregulated in the AR mice compared with the control mice (Figure 4), indicating an elevated expression of CD226 on the ILC2s of the allergic nasal mucosa.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63525/63525fig01.jpg" /> 
Figure 1: OVA-induced murine AR model. (A) The schematic diagram of the murine AR model establishment. The frequency of nasal rubbing (B) and sneezing (C) in a 10 min period. Values are presented as mean &#177; standard deviation (SD) (n = 3). For comparing the two groups, Student&#39;s t-test was performed; P &#60; 0.05 was considered statistically significant. <a href="https://www.jove.com/files/ftp_upload/63525/63525fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63525/63525fig02.jpg" /> 
Figure 2: FCM gating strategy for ILC2s. MNCs were isolated as described above. (A) The density graphic demonstrates the distribution of isolated MNCs along the FSC and SSC axes. (B) Singlets were gated based on FSC characteristics. (C) Lin&#8722;&#160;cells were defined as CD11b, CD11c, CD3, and B220 negative expression to exclude the myeloid cells, B cells, and T cells in MNCs. Dead cells were excluded by means of fixable viability dye (FVD) staining. (D) ILCs are characterized by Lin&#8722; CD45+. (E) ILC2s are gated by CD90.2+ KLRG1+ among ILCs. <a href="https://www.jove.com/files/ftp_upload/63525/63525fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63525/63525fig03.jpg" /> 
Figure 3: ILC2s in the nasal mucosa of healthy and AR mice. (A) Representative FCM images illustrate the proportions of ILC2s among ILCs from the nasal cavity mucosa of healthy control (HC) and AR groups. (B) The proportions of ILC2s in Lin&#8722; CD45+ cells in HC and AR groups. Values are presented as the mean &#177; SD (n = 3). Student&#39;s t-test was performed for a two-group comparison. P &#60; 0.05 was considered statistically significant. <a href="https://www.jove.com/files/ftp_upload/63525/63525fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63525/63525fig04.jpg" /> 
Figure 4: CD226 expression in nasal mucosal ILC2s. ILC2s were characterized using the above-mentioned strategy. The expression of CD226 on ILC2s was evaluated using FCM. (A) Representative histograms of CD226 expression in HC and AR groups; blue area represents the CD226 FMO control. (B,C) The proportions of CD226-positve ILC2s and mean fluorescence intensity (MFI) of CD226 in the HC group and AR group. Values are expressed as the mean &#177; SD (n = 3). Student&#39;s t-test was performed for comparison between the two groups. P &#60; 0.05 was considered significant. <a href="https://www.jove.com/files/ftp_upload/63525/63525fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antibody</td>
			<td>Fluorochrome</td>
			<td>Clone</td>
			<td>Dilution</td>
		</tr>
		<tr>
			<td>CD11b</td>
			<td>FITC</td>
			<td>M1/70</td>
			<td>1:100</td>
		</tr>
		<tr>
			<td>CD11c</td>
			<td>FITC</td>
			<td>N418</td>
			<td>1:200</td>
		</tr>
		<tr>
			<td>CD226</td>
			<td>PE/Cyanine7</td>
			<td>10E5</td>
			<td>1:50</td>
		</tr>
		<tr>
			<td>CD3e</td>
			<td>FITC</td>
			<td>145-2C11</td>
			<td>1:100</td>
		</tr>
		<tr>
			<td>CD45</td>
			<td>Alexa Fluor 700</td>
			<td>30-F11</td>
			<td>1:200</td>
		</tr>
		<tr>
			<td>CD45R(B220)</td>
			<td>FITC</td>
			<td>RA3-6B2</td>
			<td>1:100</td>
		</tr>
		<tr>
			<td>CD90.2</td>
			<td>PE</td>
			<td>30-H12</td>
			<td>1:100</td>
		</tr>
		<tr>
			<td>KLRG1</td>
			<td>APC</td>
			<td>2F1</td>
			<td>1:160</td>
		</tr>
	</tbody>
</table>
Table 1: Surface staining antibody cocktail.

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

Universidad Científica del Sur

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JoVE Journal

Immunology and Infection

1.9K 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 of CD4+ T cells from Mouse Lymph Nodes Using Miltenyi MACS Purification

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several organisms including humans, non-human primates, rat and, as we describe here, mice. Magnetic bead-based cell separation allows for either positive selection (or cell depletion) as well as negative selection. Here, we demonstrate negative selection of untouched or na ve CD4+ helper T cells. Using this standard protocol we typically purify cells that are &#8805; 96% pure CD4+/CD3+. This protocol is used in conjunction with the protocol Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice published in issue 7 of JoVE, for purification of T cells and other cell types to adoptively transfer for imaging purposes. Although we did not demonstrate FACS analysis in this protocol video, it is highly recommended to check the overall purity of isolated cells using the appropriate antibodies via FACS. In addition, we demonstrate the non-sterile method of T cell isolation. If sterile cells are needed for your particular end-user application, be sure to do all of the demonstrated procedures in the tissue culture hood under standard sterile conditions. Thank you for watching and good luck with your own experiments!

Isolation of lymphocytes using the Miltenyi MACs kit is a reliable way to purify cells from whole lymphoid tissue homogenates. Cells purified using the Miltenyi system are typically &#8805; 96% pure. Here, we demonstrate the steps taken to isolate CD4+ T cells, one of the many kits offered by Miltenyi.

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several ...

Biology

Isolation of Mouse Lung Dendritic Cells

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue (NALT) in Mouse Responses to Vaccines

The nasopharyngeal-associated lymphoreticular tissues (NALT) found in humans, rodents, and other mammals, contribute to immunity in the nasal sinuses1-3. The NALT are two parallel bell-shaped structures located in the nasal passages above the hard palate, and are usually considered to be secondary components of the mucosal-associated lymphoid system4-6. Located within the NALT are discrete compartments of B and T lymphocytes interspersed with antigen-presenting dendritic cells4,7,8. These cells are surrounded by an epithelial cell layer intercalated with M-cells that are responsible for antigen retrieval from the mucosal surfaces of the air passages9,10. Naive lymphocytes circulating through the NALT are poised to respond to first encounters with respiratory pathogens7. While NALT disappear in humans by the age of two years, the Waldeyer's Ring and similarly structured lymphatic organs continue to persist throughout life6. In contrast to humans, mice retain NALT throughout life, thus providing a convenient animal model for the study of immune responses originating within the nasal sinuses11.
Cultures of single-cell suspensions of NALT are not practical due to low yields of mononuclear cells. However, NALT biology can be examined by ex vivo culturing of the intact organ, and this method has the additional advantage of maintaining the natural tissue structure. For in vivo studies, genetic knockout models presenting defects limited to NALT are not currently available due to a poor understanding of the developmental pathway. For example, while lymphotoxin-&alpha; knockout mice have atrophied NALT, the Peyer's patches, peripheral lymph nodes, follicular dendritic cells and other lymphoid tissues are also altered in these genetically manipulated mice12,13. As an alternative to gene knockout mice, surgical ablation permanently eliminates NALT from the nasal passage without affecting other tissues. The resulting mouse model has been used to establish relationships between NALT and immune responses to vaccines1,3. Serial collection of serum, saliva, nasal washes and vaginal secretions is necessary for establishing the basis of host responses to vaccination, while immune responses originating directly from NALT can be confirmed by tissue culture. The following procedures outline the surgeries, tissue culture and sample collection necessary to examine local and systemic humoral immune responses to intranasal (IN) vaccination.

Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue NALT in Mouse Responses to Vaccines

Methods to examine contributions of the nasopharyngeal-associated lymphoreticular tissues (NALT) to nasal and systemic immune responses of mice to intranasal vaccines are described. We demonstrate a surgical procedure to establish a NALT-dependent mouse model and ex vivo cultures of extracted NALT.

The nasopharyngeal-associated lymphoreticular tissues (NALT) found in humans, rodents, and other mammals, contribute to immunity in the nasal sinuses1-3. ...

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 ...

Isolation of Murine Lymph Node Stromal Cells

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host. 
Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.

Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells.

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and ...

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells are therefore characterized as an innate T cell population capable of activating and steering adaptive immune responses. The development of improved techniques for the culture and expansion of murine iNKT cells facilitates the study of iNKT cell biology in in vitro and in vivo model systems. Here we describe an optimized procedure for the isolation and expansion of murine splenic iNKT cells.
Spleens from C57Bl/6 mice are removed, dissected and strained and the resulting cellular suspension is layered over density gradient media. Following centrifugation, splenic mononuclear cells (MNCs) are collected and CD5-positive (CD5+) lymphocytes are enriched for using magnetic beads. iNKT cells within the CD5+ fraction are subsequently stained with &#945;GalCer-loaded CD1d tetramer and purified by fluorescence activated cell sorting (FACS). FACS sorted iNKT cells are then initially cultured in vitro using a combination of recombinant murine cytokines and plate-bound T cell receptor (TCR) stimuli before being expanded in the presence of murine recombinant IL-7. Using this technique, approximately 108 iNKT cells can be generated within 18-20 days of culture, after which they can be used for functional assays in vitro, or for in vivo transfer experiments in mice.

Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents.

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly motile parasite not only reaches the liver to invade hepatocytes and transform into erythrocyte-infective form. It also migrates into the skin and to the proximal lymph node draining the injection site, where it can be recognized and degraded by resident and/or recruited myeloid cells. Intravital imaging reported the early recruitment of brightly fluorescent Lys-GFP positive leukocytes in the skin and the interactions between sporozoites and CD11c+ cells in the draining lymph node. We present here an efficient procedure to recover, identify and enumerate the myeloid cell subsets that are recruited to the mouse skin and draining lymph node following intradermal injection of immunizing doses of sporozoites in a murine model. Phenotypic characterization using multi-parametric flow cytometry provides a reliable assay to assess early dynamic cellular changes during inflammatory response to Plasmodium infection.

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

We describe here a protocol for isolating myeloid cells from mouse skin and draining lymph node following intradermal injection of Plasmodium sporozoites. Flow cytometry of collected cells provides a reliable assay to characterize the skin and draining lymph node inflammatory response to the parasite.

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

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

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

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

Isolation of Tonsillar Mononuclear Cells to Study Ex Vivo Innate Immune Responses in a Human Mucosal Lymphoid Tissue

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal immunity, because they can model host-pathogen interactions in the tissue. While isolated cells derived from tissues were the first cell culture model, their use has been neglected because tissue can be hard to obtain. In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells (TMCs) from healthy human tonsils to study innate immune responses upon activation, mimicking viral infection in mucosal tissues. Isolation of TMCs from the tonsils is quick, because the tonsils barely have any epithelium and yield up to billions of all major immune cell types. This method allows detection of cytokine production using several techniques, including immunoassays, qPCR, microscopy, flow cytometry, etc., similar to the use of peripheral mononuclear cells (PBMCs) from blood. Furthermore, TMCs show a higher sensitivity to drug testing than PBMCs, which needs to be considered for future toxicity assays. Thus, ex vivo TMCs cultures are an easy and accessible mucosal model.

In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells from healthy humans undergoing partial surgical tonsillectomy to study innate immune responses upon activation, mimicking viral infection in mucosal tissues.

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal ...

A Protocol to Isolate, Culture, and Identify Human Limbal Niche Cells

Isolation and Identification of Limbal Niche Cells

Here we report a standard procedure for the isolation and identification of limbal niche cells (LNCs). Limbus tissue obtained from an eye bank was used for LNCs isolation. The tissue was divided into 12 pieces under aseptic conditions and digested for 18 h at 37 &#176;C in the cell culture incubator using collagenase A to obtain cell clusters with LNCs and limbal&#160;epithelial progenitor cells. The cell clusters were further digested for 15 min at 37 &#176;C using 0.25% trypsin-EDTA to obtain single cells and then cultured in modified embryonic stem cell medium (MESCM) on a plastic surface coated with 5% Matrigel. Cells were passaged upon 70% confluence, and LNCs were identified using immunofluorescence, real-time quantitative PCR (qPCR), and flow cytometry. Primary LNCs were isolated and passaged more than 12 times. The proliferation activity of LNCs from P4 to P6 was the highest. LNCs expressed higher stem cell markers than BMMSCs (SCF, Nestin, Rex1, SSEA4, CD73, CD90, MSX1, P75NTR, and PDGFR&#946;). Furthermore, results showed that P4 LNCs uniformly expressed VIM, CD90, CD105, and PDGFR&#946;, but not Pan-CK, which could be used as a marker for the identification of LNCs. Flow cytometric analysis showed that approximately 95%, 97%, 92%, and 11% of LNCs expressed CD73, CD90, CD105, and SCF respectively, while they were&#160;68%, 99%, 20%, and 3% in&#160;BMMSCs. The standard process for LNC&#160;isolation and identification could provide&#160;a reliable laboratory basis for the widespread use of LNCs.

Author Spotlight: Standardizing Limbal Niche Cell (LNC) Isolation and Characterization to Support Widespread LNC Research 

Here, we present a protocol to isolate and identify the human limbal niche cells.

Here we report a standard procedure for the isolation and identification of limbal niche cells (LNCs). Limbus tissue obtained from an eye bank was used ...

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

Summary

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Isolation of Mouse Lung Dendritic Cells

Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue (NALT) in Mouse Responses to Vaccines

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Isolation of Murine Lymph Node Stromal Cells

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

Isolation of Tonsillar Mononuclear Cells to Study Ex Vivo Innate Immune Responses in a Human Mucosal Lymphoid Tissue

Isolation and Identification of Limbal Niche Cells