Name: flow cytometric analysis for identification of the innate and adaptive immune cells of murine lung
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Description: Watch this Scientific Journal Video about Flow Cytometric Analysis for Identification of the Innate and Adaptive Immune Cells of Murine Lung at JoVE.com

This work was supported by NIH grants R01CA238263 and R01CA229784 (VAB).

All studies and experiments described in this protocol were conducted under guidelines according to the Institutional Animal Care and Use Committee (IACUC) of Beth Israel Deaconess Medical Center. Six to ten weeks old C57BL/6 mice of either sex were used to develop this protocol.
1. Surgical excision and tissue preparation
<ol>
	<li>Euthanize the mouse by intraperitoneally injecting 1 mL of tribromoethanol (prepared according to standard protocol; Table of Materials). 
...

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+origin+of+lung+macrophage+diversity.">Developmental origin of lung macrophage diversity.</a> Development. 143 (8), 1318-1327 (2016).</li><li>Schyns, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-classical+tissue+monocytes+and+two+functionally+distinct+populations+of+interstitial+macrophages+populate+the+mouse+lung.">Non-classical tissue monocytes and two functionally distinct populations of interstitial macrophages populate the mouse lung.</a> Nature Communications. 10 (1), 3964 (2019).</li><li>Gibbings, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three+unique+interstitial+macrophages+in+the+murine+lung+at+steady+state.">Three unique interstitial macrophages in the murine lung at steady state.</a> American Journal of Respiratory Cell and Molecular Biology. 57 (1), 66-76 (2017).</li><li>Ural, B. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+nerve-associated,+lung-resident+interstitial+macrophage+subset+with+distinct+localization+and+immunoregulatory+properties.">Identification of a nerve-associated, lung-resident interstitial macrophage subset with distinct localization and immunoregulatory properties.</a> Science Immunology. 5 (45), 8756 (2020).</li><li>Chakarov, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+distinct+interstitial+macrophage+populations+coexist+across+tissues+in+specific+subtissular+niches.">Two distinct interstitial macrophage populations coexist across tissues in specific subtissular niches.</a> Science. 363 (6432), (2019).</li><li>Liegeois, M., Legrand, C., Desmet, C. 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Identification of pulmonary immune cells can be challenging because of the multiple immune cell types residing in the lung and their unique immunophenotypic characteristics compared to their counterparts residing in other tissues. In several pathologic conditions, cells with distinct phenotypic features appear in the lungs. For example, bleomycin-induced lung injury results in the recruitment of circulating monocyte-derived macrophages in the alveolar space, where they can remain for as long as one year and even persist ...

The murine respiratory tract contains a unique immune system responsible for fighting pathogens and maintaining immune homeostasis. The pulmonary immune system consists of cellular populations with significant heterogeneity in their phenotype, function, origin, and location. Resident alveolar macrophages (AMs), originated mainly from fetal monocytes, reside in the alveolar lumen1, while bone marrow-derived interstitial macrophages (IMs) reside in the lung parenchyma2. IMs can be further subclassified by the expression of CD206. CD206+ IMs populate the peribronchial and perivascular area, while CD206- IMs are located at the alveolar interstitium3. A few subclassifications of IMs have been proposed recently3,4,5,6. Although IMs are less studied than AMs, recent evidence supports their crucial role in the regulation of the immune system of the lung7. In addition, CD206 is also expressed in alternatively activated AMs8.
Pulmonary dendritic cells (DCs) are another heterogeneous group of lung immune cells with respect to their functional properties, location, and origin. Four subcategories of DCs have been described in the lung: conventional CD103+ DCs (also known as cDC1), conventional CD11b+ DCs (also known as cDC2), monocyte-derived DCs (MoDCs), and plasmacytoid DCs9,10,11,12,13. The first three subclasses can be defined as major histocompatibility complex (MHC) II+CD11c+9,10,14,15. Plasmacytoid DCs express MHC II and are intermediately positive for CD11c but express high levels of B220 and PDCA-19,13,16. In na&#239;ve murine lungs, CD103 DCs and CD11b DCs are located in the airway interstitium, whereas plasmacytoid DCs are located in the alveolar interstitium17.
Two major populations of monocytes reside in the lung during steady state: classical monocytes and non-classical monocytes. Classical monocytes are Ly6C+ and are critical for the initial inflammatory response. In contrast, non-classical monocytes are Ly6C- and have been widely viewed as anti-inflammatory cells3,16,18. Recently, an additional population of CD64+CD16.2+ monocytes was described, which originate from Ly6C- monocytes and give rise to CD206+ IMs3.
Eosinophils mainly appear in the lungs during helminth infection or allergic conditions. However, there is a small number of eosinophils in the pulmonary parenchyma during steady state, known as resident eosinophils. In contrast to the resident eosinophils, inflammatory eosinophils are found in the lung interstitium and bronchoalveolar lavage (BAL). In mouse models of house dust mite (HDM), inflammatory eosinophils are recruited into the lung after antigen-mediated stimulation. It has been proposed that resident eosinophils might have a regulatory role in allergy by inhibiting T helper 2 (Th2) sensitization to HDM19.
In contrast to the rest of pulmonary myeloid cells, neutrophils express Ly6G but not CD68 and are characterized by a signature of the CD68-Ly6G+ immunophenotype16,20,21. Visualization studies have shown that during steady state, the lung reserves a pool of neutrophils in the intravascular compartment and hosts a considerable number of extravascular neutrophils22. Similar to eosinophils, neutrophils are not found in BAL at steady state; however, several forms of immune stimulation, such as LPS challenge, asthma, or pneumonia, drive neutrophils into the alveolar lumen, resulting in their presence in BAL21,22,23.
A substantial number of CD45+ cells of the lung represent natural killer (NK), T cells, and B cells and are negative for most myeloid markers24. In the lungs of na&#239;ve mice, these three cell types can be identified based on the expression of CD11b and MHC II18. Around 25% of pulmonary CD45+ cells are B cells, whereas the percentage of NK cells is higher in the lung than other lymphoid and non-lymphoid tissues24,25,26. Among pulmonary T cells, a considerable fraction is CD4-CD8- and plays an important role in respiratory infections26.
Because the lung hosts a very complex and unique immune system, several gating strategies for the identification of lung immune cells have been developed and reported16,18,20,27. The gating strategy described herein provides a comprehensive and reproducible way to identify up to 12 different pulmonary myeloid and non-myeloid immune populations using 9 markers. Additional markers have been used to validate the results. Furthermore, a detailed method is provided for the preparation of a single-cell suspension that minimizes cell death and allows the identification of the most complete profile of the immune cell compartment of the lung. It should be noted that the identification of non-immune cells of the lung, such as epithelial cells (CD45-CD326+CD31-), endothelial cells (CD45-CD326-CD31+), and fibroblasts requires a different approach28,29. Identification of such populations is not included in the protocol and method described here.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 mL syringe plunger</td>
			<td>EXELINT</td>
			<td>26265</td>
			<td></td>
		</tr>
		<tr>
			<td>18 G needles</td>
			<td>BD Precision Glide Needle</td>
			<td>305165</td>
			<td></td>
		</tr>
		<tr>
			<td>21 G needles</td>
			<td>BD Precision Glide Needle</td>
			<td>305195</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical tubes</td>
			<td>Falcon</td>
			<td>3520</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#956;m cell strainer</td>
			<td>ThermoFisher</td>
			<td>22363548</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plates</td>
			<td>Falcon/corning</td>
			<td>3799</td>
			<td></td>
		</tr>
		<tr>
			<td>ACK Lysing Buffer</td>
			<td>ThermoFisher</td>
			<td>A10492-01</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse CD11b</td>
			<td>Biolegend</td>
			<td>101215</td>
			<td>For details see Table 2</td>
		</tr>
		<tr>
			<td>anti-mouse CD11c</td>
			<td>Biolegend</td>
			<td>117339 / 117337</td>
			<td>For details see Table 2</td>
		</tr>
		<tr>
			<td>anti-mouse CD45</td>
			<td>Biolegend</td>
			<td>103115</td>
			<td>For details see Table 2</td>
		</tr>
		<tr>
			<td>anti-mouse CD64</td>
			<td>Biolegend</td>
			<td>139319</td>
			<td>For details see Table 2</td>
		</tr>
		<tr>
			<td>anti-mouse CD68</td>
			<td>Biolegend</td>
			<td>137009</td>
			<td>For details see Table 2</td>
		</tr>
		<tr>
			<td>anti-mouse GR-1</td>
			<td>Biolegend</td>
			<td>108433</td>
			<td>For details see Table 2</td>
		</tr>
		<tr>
			<td>anti-mouse Siglec F</td>
			<td>Biolegend</td>
			<td>155503</td>
			<td>For details see Table 2</td>
		</tr>
		<tr>
			<td>AVERTIN</td>
			<td>Sigma-Aldrich</td>
			<td>240486</td>
			<td></td>
		</tr>
		<tr>
			<td>B220</td>
			<td>Biolegend</td>
			<td>103228</td>
			<td>For details see Table 2</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>9048-46-8</td>
			<td></td>
		</tr>
		<tr>
			<td>CD103</td>
			<td>Biolegend</td>
			<td>121405 / 121419</td>
			<td>For details see Table 2</td>
		</tr>
		<tr>
			<td>CD24</td>
			<td>Biolegend</td>
			<td>138503</td>
			<td>For details see Table 2</td>
		</tr>
		<tr>
			<td>CD3</td>
			<td>Biolegend</td>
			<td>100205</td>
			<td>For details see Table 2</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase Type 1</td>
			<td>Worthington Biochemical Corp</td>
			<td>LS004196</td>
			<td></td>
		</tr>
		<tr>
			<td>CX3CR1</td>
			<td>Biolegend</td>
			<td>149005</td>
			<td>For details see Table 2</td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Millipore Sigma</td>
			<td>10104159001</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>F4/80</td>
			<td>Biolegend</td>
			<td>123133</td>
			<td>For details see Table 2</td>
		</tr>
		<tr>
			<td>FcBlock (CD16/32)</td>
			<td>Biolegend</td>
			<td>101301</td>
			<td>For details see Table 2</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>R&#38;D Systems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Serrated Forceps</td>
			<td>Roboz Surgical Instrument Co</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Foxp3 / Transcription Factor Staining Buffer Set</td>
			<td>ThermoFisher</td>
			<td>00-5523-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Futura Safety Scalpel</td>
			<td>Merit Medical Systems</td>
			<td>SMS210</td>
			<td></td>
		</tr>
		<tr>
			<td>Live/Dead Fixable Far Read Dead Cell Stain Kit</td>
			<td>ThermoFisher</td>
			<td>L34973</td>
			<td>For details see Table 2</td>
		</tr>
		<tr>
			<td>MERTK</td>
			<td>Biolegend</td>
			<td>151505</td>
			<td>For details see Table 2</td>
		</tr>
		<tr>
			<td>MHC-II</td>
			<td>Biolegend</td>
			<td>107621</td>
			<td>For details see Table 2</td>
		</tr>
		<tr>
			<td>NK1.1</td>
			<td>Biolegend</td>
			<td>108705</td>
			<td>For details see Table 2</td>
		</tr>
		<tr>
			<td>Orbital Shaker</td>
			<td>VWR</td>
			<td>Model 200</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Falcon</td>
			<td>351029</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerated benchtop centrifuge</td>
			<td>SORVAL ST 16R</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Small curved scissor</td>
			<td>Roboz Surgical Instrument Co</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

flow cytometric analysis for identification of the innate and adaptive immune cells of murine lung

The respiratory tract is in direct contact with the outside environment and requires a precisely regulated immune system to provide protection while suppressing unwanted reactions to environmental antigens. Lungs host several populations of innate and adaptive immune cells that provide immune surveillance but also mediate protective immune responses. These cells, which keep the healthy pulmonary immune system in balance, also participate in several pathological conditions such as asthma, infections, autoimmune diseases, and cancer. Selective expression of surface and intracellular proteins provides unique immunophenotypic properties to the immune cells of the lung. Consequently, flow cytometry has an instrumental role in the identification of such cell populations during steady-state and pathological conditions. This paper presents a protocol that describes a consistent and reproducible method to identify the immune cells that reside in the lungs of healthy mice under steady-state conditions. However, this protocol can also be used to identify changes in these cell populations in various disease models to help identify disease-specific changes in the lung immune landscape.

Gating strategy 
The first step of our gating strategy is the exclusion of the debris and doublets (Figure 1A). Careful exclusion of doublets is critical to avoid false-positive populations (Supplemental&#160;Figure S2). Then, immune cells are identified using CD45+, a marker for hematopoietic cells (Figure 1B). The live-dead stain can be added to exclude dead cells. However, this protocol results in the death of...

Watch this Scientific Journal Video about Flow Cytometric Analysis for Identification of the Innate and Adaptive Immune Cells of Murine Lung at JoVE.com

Flow Cytometric Analysis for Identification of the Innate and Adaptive Immune Cells of Murine Lung

In this study, we present an effective and reproducible protocol to isolate the immune populations of the murine respiratory system. We also provide a method for the identification of all innate and adaptive immune cells that reside in the lungs of healthy mice, using a 9-color-based flow cytometry panel.

The respiratory tract is in direct contact with the outside environment and requires a precisely regulated immune system to provide protection while ...

Because the lung hosts a very complex and unique immune system, several gating strategies for identification of lung immune cells have been developed and reported. The existence of several different approaches makes it difficult to compare results generated by different laboratories. Our gating strategy provides a comprehensive and reproducible way to identify up to 12 different pulmonary myeloid and non-myeloid immune populations using nine markers.The present protocol describes characterization and identification of lung immune populations under steady-state conditions. However, this protocol can be employed to identify changes of these cell populations in various disease models, where it can help identify disease-specific changes of the lung immune landscape. Investigators who perform this technique for the first time should pay attention to the digestion conditions and reagents, as they make a big difference in the release of immune cells from the lung tissue.Begin by preparing the euthanized animal for surgery. Stabilize the mouse dorsally by using needles or tape on the four extremities, then use 70%ethanol to sanitize the skin of the ventral area. Make an incision from the neck to the abdomen.Remove the skin from the thoracic area, along with the ribs and the sternum. Flush the lungs by injecting 10 milliliters of cold PBS into the right ventricle using an 18 to 21-gauge needle until they become completely white. Then, remove the thymus and heart without touching the lungs.Detach the lungs from the surrounding tissues and transfer them into a tube containing cold BSA buffer. Transfer the lung to a Petri dish, mince it using two fine scalpels, and then place it into a 50-milliliter conical tube. Add five milliliters of digestion buffer to wash the plate.Secure the lid of the tube and digest the lung for 30 minutes on an orbital shaker at a speed of 150 rotations per minute at 37 degrees Celsius. Stop the reaction by adding 10 milliliters of cold BSA buffer. After digestion, mix and dissolve the lung pieces using an 18-gauge needle.Place a 70-micrometer filter strainer on top of a new 50-milliliter conical tube and transfer the digested lung mixture into the strainer. Use the rubber side of a 10-milliliter syringe plunger to smash the remaining lung pieces on the filter and wash it with BSA buffer. Centrifuge the single-cell suspension at 350 G for eight minutes at seven degrees Celsius.Discard the supernatant and resuspend the cells in one milliliter of ACK lysis buffer. Mix the suspension using a one-milliliter pipette and incubate it for 90 seconds at room temperature. Add 10 milliliters of cold BSA buffer to the reaction mixture and centrifuge it at 350 G for seven minutes at four degrees Celsius.Discard the supernatant, resuspend the pellet in staining buffer, and count the cells using a hemocytometer. Resuspend the cells at a concentration of 5 million cells per milliliter for surface staining. Transfer 1 million cells in 200 microliters per well into a 96-well plate and centrifuge the plate at 350 G for seven minutes at four degrees Celsius.Prepare a flow cytometry block solution by diluting anti-1632 antibody in staining buffer. Resuspend the cells in 50 microliters of the flow cytometry block solution. Incubate the suspension for 15 to 20 minutes at four degrees for Celsius or on ice.Then, add 150 microliters of staining buffer to the plate and centrifuge it 350 G for five minutes at four degrees Celsius. Prepare surface antibody solution by diluting the surface antibodies in staining buffer. Resuspend the cells in 50 microliters of the surface antibody solution and incubate the plate at four degrees Celsius in the dark.Then, wash the cells twice with staining buffer. Prepare the fixation and permeabilization buffer by mixing three parts fixation and permeabilization diluent and one part staining buffer. Resuspend the cells in 50 microliters of the preprepared buffer per well of the 96-well plate and incubate them for 20 to 25 minutes at four degrees Celsius in the dark.Dilute the permeabilization buffer with purified deionized water to prepare one times permeabilization buffer and use it to wash the cells. Prepare an intracellular antibody solution by diluting with one milliliter of permeabilization buffer. Resuspend the cells in 50 microliters of the diluted intracellular antibody solution per well of the 96-well plate and incubate for 40 minutes at four degrees Celsius in the dark.Wash the cells with permeabilization buffer, then with staining buffer. After the final wash, resuspend the cells in 200 microliters of staining buffer. Acquire a minimum of 1.5 million cells per sample on the flow cytometer.After a successful surgery and appropriate postoperative procedure, the debris and doublets were excluded through a gating strategy. Immune cells were identified using the CD45+hematopoietic marker via flow cytometry. The cells were distinguished as live or dead.The immune lung cells were categorized into three groups using anti-GR-1 and anti-CD68 antibodies. Neutrophils were identified and verified using Ly6G as a unique marker. The CD45+population also contains natural killer cells, T cells, and B cells.These cells were stained and verified by natural killer 1.1, CD3, and B220 antibodies. The bronchoalveolar lavage identified eosinophils which were positive for the CD11b marker, and alveolar macrophages that did not express high levels of CD11b. CD45+dendritic cells were found and confirmed by CD24 expression.These cells showed either a positive or negative response towards CD103 and CD11b markers. CD103 negative cells were classified as conventional and monocyte-derived dendritic cells based on CD64 expression. the monocyte-derived dendritic cells were low positive for the CD24 dendritic marker, and positive for the CD64 pan-macrophage marker.Interstitial macrophages with classical and non-classical monocytes were distinguished based on CD64 and GR-1 expression. These cells showed positive expression for CX3C chemokine receptor one. The most important steps in this procedure are proper tissue harvesting, digestion, and preparation of single-cell suspension, and gating on live cells and singlets.In addition to characterizing immune cells of the lung by flow cytometry, cell culture and functional assays can be performed in isolated cell populations. This technique can pave the way for researchers to explore new questions in diseases of the lung, such as infections, autoimmune diseases, and cancer.

Research

JoVE Journal

Immunology and Infection

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

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 ...

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 ...

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both ...