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). 
	NOTE: CO2 asphyxiation should be avoided in lung studies as it might cause lung injury and alter the features and properties of lung immune cells. Cervical dislocation should also be avoided as it might cause mechanical injury of the lung.</li>
	<li>Transfer the mouse to a clean and dedicated area for surgical operation.</li>
	<li>Stabilize the mouse dorsal side down by using needles or tape on the four extremities. Use 70% ethanol to sanitize the skin of the ventral area.</li>
	<li>Perform an incision in the skin, from the neck to the abdomen. Carefully remove the skin from the thoracic area.</li>
	<li>Carefully remove the sternum and ribs.</li>
	<li>Flush the lungs by injecting 10 mL of cold PBS directly in the right ventricle, using an 18-21 G needle, until the lungs become completely white.</li>
	<li>Carefully remove the thymus and heart without touching the lungs.</li>
	<li>Gently detach the lungs from the surrounding tissues and transfer them to a tube with cold BSA buffer (Table 1). 
	NOTE: Effort should be made to remove all adjacent fat from the lungs before further preparing the single-cell suspension, as this could bias the readouts.</li>
</ol>
2. Preparation of single-cell suspension
<ol>
	<li>Transfer the lungs to an empty Petri dish and mince them with two fine scalpels. Transfer all the pieces of the minced lung to a new 50 mL conical tube. Use 5 mL of digestion buffer to wash the plate and add it to the 50 mL tube containing the minced lung (Table 1). 
	NOTE: Digestion buffer should be prepared immediately before use. Use 5 mg/mL of collagenase28. Combining 1 or 5 mg of collagenase with BSA buffer or protein-free PBS did not improve results (Supplemental Figure S1).</li>
	<li>Secure the lid of the tube and digest the lung for 30 min on an orbital shaker at a speed of 150 rpm at 37 &#176;C. Stop the reaction by adding 10 mL of cold BSA buffer.</li>
	<li>After digestion, use an 18 G needle to mix and dissolve the lung pieces. Place a 70 &#181;m filter strainer at the top of a new 50 mL conical tube. 
	NOTE: Usage of a smaller micron filter might result in the loss of major myeloid populations.</li>
	<li>Slowly transfer the digested lung mixture directly on the strainer. Use the rubber side of a 10 mL syringe plunger to smash the remaining lung pieces on the filter. Wash the processed material on the filter with BSA buffer.</li>
	<li>Centrifuge the single-cell suspension at 350 &#215; g for 8 min at 4 &#176;C.</li>
	<li>Carefully discard the supernatant and resuspend the cells in 1 mL of ACK lysis buffer. Mix well using a 1 mL pipet, and incubate for 90 s at room temperature.</li>
	<li>Add 10 mL of cold BSA buffer to stop the reaction and centrifuge at 350 &#215; g for 7 min at 4 &#176;C.Carefully discard the supernatant and resuspend the pellet in Staining Buffer to count the cells using a hemocytometer.</li>
	<li>Resuspend the cells at a concentration of 5 &#215; 106 cells/mL and use them for surface staining (see section 3). 
	NOTE: For this purpose, plate the cells in a 96-well round-bottom plate followed by antibody staining and washes. If a plate centrifuge is not available, use flow tubes instead of plates. With this protocol, ~15-20 &#215; 106 cells per lung can be obtained from a 6-10-week-old C57BL/6 mouse of average size.</li>
</ol>
3. Surface antibody staining
<ol>
	<li>Transfer 1 &#215; 106 cells in 200 &#181;L per well in a 96-well plate. Centrifuge the plate at 350 &#215; g for 7 min at 4 &#176;C. In the meantime, prepare the Fc-block solution by diluting anti-16/32 antibody (1:100) in staining buffer (Table 1).</li>
	<li>Resuspend the cells in 50 &#181;L of the pre-prepared Fc-blocking solution (Table of Materials) and incubate for 15-20 min at 4 &#176;C or on ice.</li>
	<li>Add 150 &#181;L of staining buffer and centrifuge the plate at 350 &#215; g for 5 min at 4 &#176;C. Meanwhile, prepare the surface antibody cocktail by diluting surface antibodies (1:100; Table 2) in staining buffer. 
	NOTE: (i) Anti-16/32 antibody for Fc-blocking can be used with the surface antibodies in the same mixture. (ii) If fixable viability dye is used, add it to the surface antibody cocktail at a dilution of 1:1,000.</li>
	<li>Resuspend the cells in 50 &#181;L of the pre-prepared surface antibody cocktail and incubate for 30-40 min at 4 &#176;C in the dark. Wash the cells with staining buffer twice. 
	&#8203;NOTE: If no intracellular staining is required, resuspend the cells in 200 &#181;L of staining buffer and proceed directly to the acquisition of data on the flow cytometer. Alternatively, cells might be fixed and stored at 4 &#176;C for acquisition later. We recommend using the cells for flow cytometry within 24 h.</li>
</ol>
4. Cell fixation and intracellular staining
<ol>
	<li>Prepare the fixation/permeabilization buffer (Fix/Perm Buffer) by mixing three parts of fixation/permeabilization concentrate and 1 part of fixation/permeabilization diluent of the FoxP3/Transcription Factor Staining Buffer Set (Table 1).</li>
	<li>Resuspend the cells in 50 &#181;L of the pre-prepared Fix/Perm Buffer per well of the 96-well plate, where cells were plated as described in section 3, and incubate them for 20-25 min at 4 &#176;C in the dark.</li>
	<li>Dilute the 10x permeabilization buffer as 1: 10 in purified deionized water to prepare 1x permeabilization buffer.</li>
	<li>Wash the cells once with 1x permeabilization buffer. Meanwhile, prepare the intracellular antibody cocktail by diluting intracellular antibodies (1:100) in 1 mL of permeabilization buffer.</li>
	<li>Resuspend the cells using 50 &#181;L of the pre-prepared surface antibody cocktail per cell of the 96-well plate and incubate for 40 min at 4 &#176;C in the dark.</li>
	<li>Wash the cells once with permeabilization buffer and once with staining buffer. After the final wash, resuspend the cells in 200 &#181;L of staining buffer. 
	NOTE: If no flow cytometer with plate reader is available, transfer the cells into flow cytometry tubes.</li>
	<li>Acquire a minimum of 1.5 &#215; 106 cells per sample on the flow cytometer. 
	NOTE: For single colors and unstained control samples, 0.5-1 &#215; 106 cells per sample will be sufficient. It is recommended to titer the individual antibodies used to achieve optimal staining and reduce costs. The present protocol has been optimized using Fix/Perm Buffer prepared using the FoxP3 staining buffer set. Because CD68 is a cytoplasmic and not a nuclear marker, other permeabilization solutions such as a low concentration of paraformaldehyde or cytofix/ cytoperm kits from various vendors might be sufficient.</li>
</ol>

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V.A.B. has patents on the PD-1 pathway licensed by Bristol-Myers Squibb, Roche, Merck, EMD-Serono, Boehringer Ingelheim, AstraZeneca, Novartis, and Dako. The authors declare no other competing financial interests.

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><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 &amp;mu;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&amp;amp;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 ...

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Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

6.4K Views.  Beth Israel Deaconess Medical Center, Harvard Medical School. 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.

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

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 ...

Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in peripheral tissues in steady state; however in response to antigen stimulation, DCs take up the antigen and rapidly migrate to the draining lymph nodes where they initiate T cell response against the antigen1,2. Additionally, DCs also play a key role in initiating autoimmune as well as allergic immune response3.
DCs play an essential role in both initiation of immune response and induction of tolerance in the setting of lung environment4. Lung environment is largely tolerogenic, owing to the exposure to vast array of environmental antigens5. However, in some individuals there is a break in tolerance, which leads to induction of allergy and asthma. In this study, we describe a strategy, which can be used to monitor airway DC maturation and migration in response to the antigen used for sensitization. The measurement of airway DC maturation and migration allows for assessment of the kinetics of immune response during airway allergic inflammation and also assists in understanding the magnitude of the subsequent immune response along with the underlying mechanisms.
Our strategy is based on the use of ovalbumin as a sensitizing agent. Ovalbumin-induced allergic asthma is a widely used model to reproduce the airway eosinophilia, pulmonary inflammation and elevated IgE levels found during asthma6,7. After sensitization, mice are challenged by intranasal delivery of FITC labeled ovalbumin, which allows for specific labeling of airway DCs which uptake ovalbumin. Next, using several DC specific markers, we can assess the maturation of these DCs and can also assess their migration to the draining lymph nodes by employing flow cytometry.

We describe a strategy to monitor maturation and migration of pulmonary dendritic cells in response to ovalbumin in the setting of ovalbumin induced allergic airway inflammation. This strategy can be modified to assess migration of pulmonary dendritic cells in settings of infection.

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in ...

Human Lung Dendritic Cells: Spatial Distribution and Phenotypic Identification in Endobronchial Biopsies Using Immunohistochemistry and Flow Cytometry

The lungs are constantly exposed to the external environment, which in addition to harmless particles, also contains pathogens, allergens, and toxins. In order to maintain tolerance or to induce an immune response, the immune system must appropriately handle inhaled antigens. Lung dendritic cells (DCs) are essential in maintaining a delicate balance to initiate immunity when required without causing collateral damage to the lungs due to an exaggerated inflammatory response. While there is a detailed understanding of the phenotype and function of immune cells such as DCs in human blood, the knowledge of these cells in less accessible tissues, such as the lungs, is much more limited, since studies of human lung tissue samples, especially from healthy individuals, are scarce. This work presents a strategy to generate detailed spatial and phenotypic characterization of lung tissue resident DCs in healthy humans that undergo a bronchoscopy for the sampling of endobronchial biopsies. Several small biopsies can be collected from each individual and can be subsequently embedded for ultrafine sectioning or enzymatically digested for advanced flow cytometric analysis. The outlined protocols have been optimized to yield maximum information from small tissue samples that, under steady-state conditions, contain only a low frequency of DCs. While the present work focuses on DCs, the methods described can directly be expanded to include other (immune) cells of interest found in mucosal lung tissue. Furthermore, the protocols are also directly applicable to samples obtained from patients suffering from pulmonary diseases where bronchoscopy is part of establishing the diagnosis, such as chronic obstructive pulmonary disease (COPD), sarcoidosis, or lung cancer.

Lung-resident immune cells, including dendritic cells (DCs) in humans, are critical for defense against inhaled pathogens and allergens. However, due to the scarcity of human lung tissue, studies are limited. This work presents protocols to process human mucosal endobronchial biopsies for studying lung DCs using immunohistochemistry and flow cytometry.

The lungs are constantly exposed to the external environment, which in addition to harmless particles, also contains pathogens, allergens, and toxins. In ...

Bronchoalveolar Lavage of Murine Lungs to Analyze Inflammatory Cell Infiltration

Bronchoalveolar Lavage (BAL) is an experimental procedure that is used to examine the cellular and acellular content of the lung lumen ex vivo to gain insight into an ongoing disease state.
Here, a simple and efficient method is described to perform BAL on murine lungs without the need of special tools or equipment. BAL fluid is isolated by inserting a catheter in the trachea of terminally anesthetized mice, through which a saline solution is instilled into the bronchioles. The instilled fluid is gently retracted to maximize BAL fluid retrieval and to minimize shearing forces. This technique allows the viability, function, and structure of cells within the airways and BAL fluid to be preserved.
Numerous techniques may be applied to gain further understanding of the disease state of the lung. Here, a commonly used technique for the identification and enumeration of different types of immune cells is described, where&#160;flow cytometry is combined with a select panel of fluorescently labeled cell surface-specific markers. The BAL procedure presented here can also be used to analyze infectious agents, fluid constituents, or inhaled particles within murine lungs.

The health status of the lung is reflected by the type and number of immune cells that are present in the bronchioles of the lung. We describe a bronchoalveolar lavage technique that allows the isolation and study of nonadherent cells and soluble factors from the lower respiratory tract of mice.

Bronchoalveolar Lavage (BAL) is an experimental procedure that is used to examine the cellular and acellular content of the lung lumen ex vivo to gain ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Characterization of Immune Cells and Proinflammatory Mediators in the Pulmonary Environment

Immune cell expansion, activation, and trafficking to the lungs, which are controlled by the expression of multiple cytokines and chemokines, may be altered by severe brain injury. This is evidenced by the fact that pneumonia is a major cause of mortality in patients who have suffered from ischemic stroke. The goal of this protocol is to describe the use of multicolor flow cytometric analysis to identify 13 types of immune cells in the lungs of mice, including alveolar macrophages, interstitial macrophages, CD103+ or CD11b+ dendritic cells (DCs), plasmacytoid DCs, eosinophils, monocytes/monocyte-derived cells, neutrophils, lymphoid-derived&#160; T and B cells, NK cells, and NKT cells, following ischemic stroke induction by transient middle cerebral artery occlusion. Moreover, we describe the preparation of lung homogenates using a bead homogenization method, to determine the expression levels of 13 different cytokines or chemokines simultaneously by multiplex bead arrays coupled with flow cytometric analysis. This protocol can also be used to investigate the pulmonary immune response in other disease settings, such as infectious lung disease or allergic disease.

This protocol describes the use of flow cytometry to identify the changes in immune cell composition, cytokine profile, and chemokine profile in the pulmonary environment following transient middle cerebral artery occlusion, a murine model of ischemic stroke.

Immune cell expansion, activation, and trafficking to the lungs, which are controlled by the expression of multiple cytokines and chemokines, may be ...

Assessing Respiratory Immune Responses to Haemophilus Influenzae

Haemophilus influenzae (Hi) is a prevalent bacterium found in a range of respiratory conditions. A variety of different assays/techniques may be used to assess the respiratory immune/inflammatory response to this bacterium. Flow cytometry and confocal microscopy are fluorescence-based technologies that allow detailed characterization of biological responses.&#160;Different forms of Hi antigen can be used, including cell wall components, killed/inactivated preparations, and live bacteria. Hi is a fastidious bacterium that requires enriched media but is generally easy to grow in standard laboratory settings. Tissue samples for stimulation with Hi may be obtained from peripheral blood, bronchoscopy, or resected lung (e.g., in patients undergoing surgery for the treatment of lung cancer). Macrophage and neutrophil function may be comprehensively assessed using flow cytometry with a variety of parameters measured, including phagocytosis, reactive oxygen species, and intracellular cytokine production. Lymphocyte function (e.g., T cell and NK cell function) may be specifically assessed using flow cytometry, principally for intracellular cytokine production. Hi infection is a potent inducer of extracellular trap production, both by neutrophils (NETs) and macrophages (METs). Confocal microscopy is arguably the most optimal way to assess NET and MET expression, which may also be used to assess protease activity. Lung immunity to Haemophilus influenzae can be assessed using flow cytometry and confocal microscopy.

Assessing Respiratory Immune Responses to Haemophilus Influenzae

Haemophilus influenzae induces inflammation in the respiratory tract. This article will focus on the use of flow cytometry and confocal microscopy to define immune responses by phagocytes and lymphocytes in response to this bacterium.

Haemophilus influenzae (Hi) is a prevalent bacterium found in a range of respiratory conditions. A variety of different assays/techniques may be used to ...

Advanced Imaging of Lung Homing Human Lymphocytes in an Experimental In Vivo Model of Allergic Inflammation Based on Light-sheet Microscopy

Overwhelming tissue accumulation of highly activated immune cells represents a hallmark of various chronic inflammatory diseases and emerged as an attractive therapeutic target in the clinical management of affected patients. In order to further optimize strategies aiming at therapeutic regulation of pathologically imbalanced tissue infiltration of pro-inflammatory immune cells, it will be of particular importance to achieve improved insights into disease- and organ-specific homing properties of peripheral lymphocytes. The here described experimental protocol allows to monitor lung accumulation of fluorescently labeled and adoptively transferred human lymphocytes in the context of papain-induced pulmonary inflammation. In contrast to standard in vitro assays frequently used for the analysis of immune cell migration and chemotaxis, the now introduced in vivo setting takes into account lung-specific aspects of tissue organization and the influence of the complex inflammatory scenario taking place in the living murine organism. Moreover, three-dimensional cross-sectional light-sheet fluorescence microscopic imaging does not only provide quantitative data on infiltrating immune cells, but also depicts the pattern of immune cell localization within the inflamed lung. Overall, we are able to introduce an innovative technique of high value for immunological research in the field of chronic inflammatory lung diseases, which can be easily applied by following the provided step-by-step protocol.

The here introduced protocol allows characterization of the lung homing capacity of primary human lymphocytes under in vivo inflammatory conditions. Pulmonary infiltration of adoptively transferred human immune cells in a mouse model of allergic inflammation can be imaged and quantified by light-sheet fluorescence microscopy of chemically cleared lung tissue.

Overwhelming tissue accumulation of highly activated immune cells represents a hallmark of various chronic inflammatory diseases and emerged as an ...

Medicine

Isolation of Immune and Non&mdash;Immune Cells from Mouse Lungs for Flow Cytometry

Isolation of Live Myeloid and Epithelial Cell Populations from the Mouse Lung

The lung is continuously exposed to pathogens and other noxious environmental stimuli, rendering it vulnerable to damage, dysfunction, and the development of disease. Studies utilizing mouse models of respiratory infection, allergy, fibrosis, and cancer have been critical to reveal mechanisms of disease progression and identify therapeutic targets. However, most studies focused on the mouse lung prioritize the isolation of either immune cells or epithelial cells, rather than both populations concurrently. Here, we describe a method for preparing a comprehensive single-cell suspension of both immune and non-immune populations suitable for flow cytometry and fluorescence-activated cell sorting. These populations include epithelial cells, endothelial cells, fibroblasts, and a variety of myeloid cell subsets. This protocol entails bronchoalveolar lavage and subsequent inflation of the lungs with dispase. Lungs are then digested in a liberase mixture. This method of processing liberates a variety of diverse cell types and results in a single-cell suspension that does not require manual dissociation against a filter, promoting cell survival and yielding high numbers of live cells for downstream analyses. In this protocol, we also define gating schemes for epithelial and myeloid cell subsets in both na&#239;ve and influenza-infected lungs. Simultaneous isolation of live immune and non-immune cells is key for interrogating intercellular crosstalk and gaining a deeper understanding of lung biology in health and disease.

This protocol details the isolation of live immune and non-immune populations from the mouse lung at a steady state and following influenza infection. It also provides gating strategies for identifying epithelial and myeloid cell subsets.

The lung is continuously exposed to pathogens and other noxious environmental stimuli, rendering it vulnerable to damage, dysfunction, and the development ...

Identification and Enumeration of Immune Cells in Bronchoalveolar Lavage Fluid by Flow Cytometry

Source: Van Hoecke, L. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/55398">Bronchoalveolar Lavage of Murine Lungs to Analyze Inflammatory Cell Infiltration.</a>&#160;J. Vis. Exp.&#160;(2017)
This video demonstrates the use of flow cytometry to identify and count various immune cells in bronchoalveolar lavage fluid. Initially, single immune cells are identified using a viability dye, followed by differential staining of surface marker proteins, enabling the precise enumeration of each cell type.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

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

Summary

Explore More Videos

Chapters in this video

Isolation of Mouse Lung Dendritic Cells

Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation

Human Lung Dendritic Cells: Spatial Distribution and Phenotypic Identification in Endobronchial Biopsies Using Immunohistochemistry and Flow Cytometry

Bronchoalveolar Lavage of Murine Lungs to Analyze Inflammatory Cell Infiltration

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Characterization of Immune Cells and Proinflammatory Mediators in the Pulmonary Environment

Assessing Respiratory Immune Responses to Haemophilus Influenzae

Advanced Imaging of Lung Homing Human Lymphocytes in an Experimental In Vivo Model of Allergic Inflammation Based on Light-sheet Microscopy

Isolation of Live Myeloid and Epithelial Cell Populations from the Mouse Lung

Identification and Enumeration of Immune Cells in Bronchoalveolar Lavage Fluid by Flow Cytometry