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 after bleomycin-induced fibrosis. In contrast to tissue-resident AMs, the circulating monocyte-derived macrophages are Siglec FlowCD11b+. Targeted depletion of the monocyte-derived macrophages results in the amelioration of bleomycin-mediated pulmonary fibrosis16,40. Cells with similar features are recruited to the lungs during influenza infection and provide prolonged protection against streptococcal pneumonia15.
Based on CD11c and MHC II expression, IMs have been further subcategorized into IM1, IM2, and IM3. IM1 are immunophenotypically defined as CD11c-MHC II-; IM2 are defined as CD11c-MHC II+; and IM3 are defined as MHC II+CD11c+. It has been proposed that IM1, IM2, and IM3 represent the physiologic stages of monocyte to macrophage transition, rather than distinct macrophages categories, with IM3 representing the monocytic compartment41. As mentioned above, MoDCs are low-positive for the DC marker CD24 and positive for pan-macrophage markers, including F4/80, CD64, and MERTK9,10,11,13,33,34. However, several studies identify CD64+MERKT+ cells as pulmonary macrophages4,10. Both IM3 and MoDCs have been defined as CD64+MERKT+MHCII+CD11C+,&#160;suggesting that these two populations most likely represent the same cell type. Consistent with this hypothesis, the gating strategy presented here does not identify a distinct population representing IM3 cells, in addition to MoDCs.
Critical steps of the protocol described here include: 1) the removal of all adjacent fat from the lungs before preparing the single-cell suspension, as this could bias the readouts; 2) permeabilization before staining with the anti-CD68 antibody. A limitation of the present protocol is that it cannot identify non-immune cells of the lung, such as epithelial (CD45-CD326+CD31-), endothelial cells (CD45-CD326-CD31+), and fibroblasts. Identification of such populations requires a different approach28,29. In addition, the protocol requires staining for CD68, an intracellular marker, which might pose a limitation if the investigator is not experienced in intracellular staining. The significance of the present protocol and gating strategy with respect to existing methods is that this strategy provides a streamlined approach that uses a lower number of markers while allowing reproducible identification of all the immune populations of the lung.
Furthermore, a detailed method is provided for the preparation of a single-cell suspension that minimizes cell death and allows the identification of a complete profile of the immune cell compartment of the lung. Although the protocol outlined here describes the characterization and identification of lung immune populations under steady-state conditions, future applications may include the assessment of these populations in various disease models where it can help identify disease-specific changes in the lung immune landscape. In conclusion, this article presents a simple and reproducible protocol for lung single-cell preparation and a 9-color-based flow cytometry panel for the identification of 12 different immune cell populations.

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 &#60;5% of the CD45+ cells (Figure 1C), whereas more CD45- cells are identified as dead (Supplemental Figure S3).
To identify monocytes and neutrophils, in contrast to previous studies that used specific antibodies for each of these populations16,18, we prefer to use an anti-GR-1 antibody that identifies both Ly6C+ and Ly6G+ cells. Using the anti-GR-1 antibody together with anti-CD68 allows the separation of the lung immune cells into three clusters: CD68-GR-1+, CD68+(that can be further identified as GR-1+ or GR-1-), and CD68-GR-1-/int (Figure 1D). CD68 is a marker that is predominantly detected intracellularly. Surface CD68 was probed but could not be detected without fixation/permeabilization (Supplemental Figure S4).
Polymorphonuclear cells (neutrophils) were identified as CD45+CD68-GR-1+CD11b+ (Figure 1E). These results were verified using GR1 together with an antibody specific for Ly6G (Figure 2), a unique marker for polymorphonuclear neutrophils16,18,27. Within the CD45+CD68-GR-1-/int population, approximately 10-20% are MHCII- and CD11blow and represent the NK cells (Figure 1F). NK1.1, a unique marker for NK cells, was used to confirm this (Figure 3)25,30. The CD45+CD68-GR1-/intCD11b- population consists of MHCII- T cells and MHCII+ B cells (Figure 1F). Two additional antibodies were used to verify T cells and B cells-CD3 and B220, respectively (Figure 3).
In healthy mice under steady-state conditions, the majority of CD45+ cells detected in BAL are AMs, the main residents of the airways. Hence, BAL was performed to assess the markers that characterize AMs20,31. These cells are CD45+CD68+Siglec-F+CD11c+ but, unlike other myeloid cells, do not express high levels of CD11b (Figure 4). The same combination of immune markers also identifies AMs in homogenates of total lungs (Figure 1G,H). In addition to AMs, in the CD45+CD68+Sigle-F+ gate (Figure 1G), there is a distinct cell population that is positive for CD11b but not for CD11c. This CD45+CD68+Siglec-F+CD11b+CD11c- population of leukocytes represents eosinophils (Figure 1G)19,32.
In the gate with the CD45+CD68+SiglecF- cells (Figure 1G), there is a CD11c+MHC+ population that represents pulmonary DCs (Figure 1I). Several investigators identify pulmonary DCs as CD11c+MHCII+CD24+16,18.&#160;CD24 expression was assessed to confirm the identity of this population (Figure 5A). The majority of the lung DCs are either CD103+CD11b- or CD103-CD11b+ (Figure 1J). The CD103+CD11b- DCs represent the CD103+ conventional DCs and are identified as CD45+CD68+SiglecF-MHCII+CH11c+CD103+CD11b-. In contrast, CD103-CD11b+ DCs are divided into conventional DCs and MoDCs based on CD64 expression (Figure 1K). Therefore, conventional CD11b+ DCs are identified as CD45+CD68+SiglecF-MHCII+CH11c+CD103-CD11b+CD64- while the MoDCs are identified as CD45+CD68+SiglecF-MHCII+CH11c+CD103-CD11b+CD64+. In contrast to conventional DCs, MoDCs are low-positive for the DC marker CD24 and positive for pan-macrophage markers, including F4/80, CD64 and MERTK (Figure 5A)9,10,11,13,33,34.
IMs and classical and non-classical monocytes are in the CD45+CD68+Siglec-F-CD11c-/intCD11b+ gate (Figure 1L) and are distinguished based on CD64 and GR-1 expression (Figure 1M). CD64, a pan-macrophage marker, is mainly expressed by IMs and AMs, as well as a subset of DCs. Classical monocytes are also known as Ly6C+ monocytes and non-classical monocytes as Ly6C- monocytes. Both monocytes and IMs are negative for Ly6G; however, classical monocytes express Ly6C and are therefore positive for GR-1. In contrast, both interstitial macrophages and non-classical monocytes are negative for GR-1 and Ly6C3,4,11,16,18,20,35,36. In addition, non-classical monocytes are CD11cint, while classical monocytes are CD11c-. Although this difference in CD11c expression is generally insignificant for distinguishing the two types of monocytes at baseline, it could be critical for pathological conditions when classical monocytes accumulate. Based on the above, interstitial macrophages are defined as CD45+CD68+Siglec-F-CD11c-/intCD11b+GR-1-CD64+, classical monocytes as CD45+CD68+Siglec-F-CD11c-CD11b+GR-1+CD64-, and non-classical monocytes as CD45+CD68+CD11c-/intCD11b+GR-1-CD64-. Both AMs and IMs are positive for all the macrophage markers, including CD68, CD64, F4/80, and MERTK proto-oncogene. However, unlike IMs, AMs are CX3CR1-, in contrast to IMs, which could be explained by the difference in the origin of the two types of lung macrophages4,37,38,39 (Figure 5B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62985/62985fig01.jpg" /> 
Figure 1: Gating strategy of immune cells present in the murine lung. After careful exclusion of debris, doublets, dead cells, and the non-immune cells (CD45-) (A-C), CD45+ cells were separated into 3 main clusters based on the expression of CD68 and GR-1 (D). Neutrophils belong to the CD68-GR-1+ population (E) while the CD68-GR-1-/int population consists of NK cells, B cells, and T cells (F). CD68+ cells can be further divided into Siglec F+ cells (G), which are AMs and eosinophils (H), and Siglec F- cells (G), which consist of monocytes, IMs, and DCs (I-M).&#160;Abbreviations: SSC-A = peak area of side-scattered light; FSC-A = peak area of forward-scattered light; SSC-H = peak height of side-scattered light; L/D = live/dead staining; MHC = major histocompatibility complex; SF = Siglec F; GR-1 = GPI-linked myeloid differentiation marker (Ly-6G); NK = natural killer; IMs = interstitial macrophages; DCs = dendritic cells; AMs = alveolar macrophages. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62985/62985fig01large.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/62985/62985fig02.jpg" /> 
Figure 2: Ly6G in the lung is expressed only by CD45+CD68-GR-1+CD11b+ cells identified as polymorphonuclear neutrophils. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62985/62985fig02large.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/62985/62985fig03.jpg" /> 
Figure 3: Verification of the identification of NK cells, T cells, and B cells by the gating strategy. Markers specific for NK 1.1, CD3, and B220 were used to verify the identification of NK cells, T cells, and B cells, respectively. It should be noted that NKT cells, if present, might fall in the CD3+ cell population within the NK cell gate, and caution is required to avoid contamination of NK with NKT cells. Abbreviations: NK = natural killer; MHC = major histocompatibility complex. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62985/62985fig03large.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/62985/62985fig04.jpg" /> 
Figure 4: The majority of the immune cells obtained by bronchoalveolar lavage in na&#239;ve mice are alveolar macrophages. Abbreviations: AMs = alveolar macrophages; DCs = dendritic cells; IM = interstitial macrophage; SSC-A = peak area of side-scattered light; FSC-A = peak area of forward-scattered light; SSC-H = peak height of side-scattered light; SF = Siglec F; GR-1 = GPI-linked myeloid differentiation marker (Ly-6G). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62985/62985fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62985/62985fig05.jpg" /> 
Figure 5: Comparison of the expression of different markers in immune cells. (A) Comparison of CD24, CD64, MERTK, F4/80, and CD103 expression in pulmonary DCs. (B) Comparison of CD68, CD64, MERTK, F4/80, CD11c, Siglec F (SF), CD11b, and CX3CR1 in pulmonary macrophages and monocytes. Abbreviations: DCs = dendritic cells; MoDCs = monocyte-derived DCs; AMs = alveolar macrophages; IMs = interstitial macrophages; SF = Siglec F; MERTK = myeloid-epithelial-reproductive tyrosine kinase. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62985/62985fig05large.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>BSA buffer</td>
			<td colspan="3">PBS + 0.5% Bovine serum albumin</td>
		</tr>
		<tr>
			<td>Digestion buffer</td>
			<td colspan="3">Prewarmed (37 &#176;C) BSA buffer + 5 mg/mL collagenase type 1 + 0.2 mg/mL DNase I</td>
		</tr>
		<tr>
			<td>Staining buffer</td>
			<td colspan="3">PBS + 2.5% FBS</td>
		</tr>
		<tr>
			<td>Fix/Perm Buffer</td>
			<td colspan="3">Three parts of fixation/permeabilization diluent and 1 part of fixation/permeabilization diluent of the Foxp3/Transcription Factor Staining Buffer Set</td>
		</tr>
		<tr>
			<td>Permeabilization buffer</td>
			<td colspan="3">10x permeabilization buffer from the Foxp3/Transcription Factor staining Buffer Set diluted 10 times in purified deionized water</td>
		</tr>
	</tbody>
</table>
Table 1: Buffers.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antigen</td>
			<td>Clone</td>
			<td>Fluorochrome</td>
			<td>Dilution</td>
			<td>Surface/intracellular</td>
		</tr>
		<tr>
			<td>CD45</td>
			<td>30-F11</td>
			<td>APC/CY7</td>
			<td>1:100</td>
			<td>surface</td>
		</tr>
		<tr>
			<td>Gr-1</td>
			<td>RB6-8C5</td>
			<td>BV421</td>
			<td>1:100</td>
			<td>surface</td>
		</tr>
		<tr>
			<td>CD68</td>
			<td>FA-11</td>
			<td>PerCPCy5.5</td>
			<td>1:100</td>
			<td>intracellular</td>
		</tr>
		<tr>
			<td>CD11b</td>
			<td>M1/70</td>
			<td>PECy7</td>
			<td>1:100</td>
			<td>surface</td>
		</tr>
		<tr>
			<td>Siglec F</td>
			<td>S17007L</td>
			<td>FITC</td>
			<td>1:100</td>
			<td>surface</td>
		</tr>
		<tr>
			<td>CD11c</td>
			<td>N418</td>
			<td>BV650 or BV510</td>
			<td>1:100</td>
			<td>surface</td>
		</tr>
		<tr>
			<td>CD64</td>
			<td>X54-5/7.1</td>
			<td>PE/Dazzle 594</td>
			<td>1:100</td>
			<td>surface</td>
		</tr>
		<tr>
			<td>MHC-II</td>
			<td>M5/114.15.2</td>
			<td>AF700</td>
			<td>1:100</td>
			<td>surface</td>
		</tr>
		<tr>
			<td>CD103</td>
			<td>2.00E+07</td>
			<td>PE / FITC</td>
			<td>1:100</td>
			<td>surface</td>
		</tr>
		<tr>
			<td>Live/Dead Fixable Far Read Dead Cell Stain Kit</td>
			<td></td>
			<td>FarRed (APC) or Aqua (BV510)</td>
			<td>1:1000</td>
			<td>surface</td>
		</tr>
		<tr>
			<td>CD3</td>
			<td>17A2</td>
			<td>PE</td>
			<td>1:100</td>
			<td>surface</td>
		</tr>
		<tr>
			<td>B220</td>
			<td>RA3-6B2</td>
			<td>AF488</td>
			<td>1:100</td>
			<td>surface</td>
		</tr>
		<tr>
			<td>NK1.1</td>
			<td>PK136</td>
			<td>FITC</td>
			<td>1:100</td>
			<td>surface</td>
		</tr>
		<tr>
			<td>CD24</td>
			<td>30-F1</td>
			<td>PE</td>
			<td>1:100</td>
			<td>surface</td>
		</tr>
		<tr>
			<td>MERTK</td>
			<td>2B10C42</td>
			<td>PE</td>
			<td>1:100</td>
			<td>surface</td>
		</tr>
		<tr>
			<td>F4/80</td>
			<td>BM8</td>
			<td>BV605</td>
			<td>1:100</td>
			<td>surface</td>
		</tr>
		<tr>
			<td>CX3CR1</td>
			<td>SA011F11</td>
			<td>PE</td>
			<td>1:100</td>
			<td>surface</td>
		</tr>
		<tr>
			<td>FcBlock (CD16/32)</td>
			<td>93</td>
			<td></td>
			<td>1:100</td>
			<td>surface</td>
		</tr>
	</tbody>
</table>
Table 2: Usage of monoclonal antibodies.
Supplemental Figure S1: The best cellular dissociation is achieved by 5 mg/mL of collagenase 1 in prewarmed PBS + 0.5% BSA. In all different conditions, 0.2 mg of DNAse I was also included. Abbreviations: BSA = bovine serum albumin; FSC-A = peak area of forward-scattered light; L/D = live/dead staining; SF = Siglec F. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62985/Supplementary_Figures_060121 1.pdf" target="_blank">Please click here to download this File.</a>
Supplemental Figure S2: Inclusion of doublets might result in false-positive populations. Abbreviation: FP = false positive. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62985/Supplementary_Figures_060121 2.pdf" target="_blank">Please click here to download this File.</a>
Supplemental Figure S3: More CD45- than CD45+ have features consistent with dead cells. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62985/Supplementary_Figures_060121 3.pdf" target="_blank">Please click here to download this File.</a>
Supplemental Figure S4: Surface staining for CD68 is not adequate to properly distinguish the individual immune cell populations of the lung. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62985/Supplementary_Figures_060121 4.pdf" target="_blank">Please click here to download this File.</a>

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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Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

6.3K 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

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 cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

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 increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

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

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

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

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

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

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

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

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

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 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the &#946;-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of&#160;how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists&#8217; discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

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 microbial and dietary. Given an increasing understanding that these luminal antigens help shape the immune response and that education of immune cells within the intestine is critical for a number of systemic diseases, there has been increased interest in characterizing the intestinal immune system. However, many published protocols are arduous and time-consuming. We present here a simplified protocol for the isolation of lymphocytes from the small-intestinal lamina propria, intraepithelial layer, and Peyer&#39;s patches that is rapid, reproducible, and does not require laborious Percoll gradients. Although the protocol focuses on the small intestine, it is also suitable for analysis of the colon. Moreover, we highlight some aspects that may need additional optimization depending on the specific scientific question. This approach results in the isolation of large numbers of viable lymphocytes that can subsequently be used for flow cytometric analysis or alternate means of characterization.

There is growing interest in the quantitative characterization of intestinal lymphocytes owing to increasing recognition that these cells play a critical role in a variety of intestinal and systemic diseases. In this protocol, we describe how to isolate single cell populations from different small-intestinal compartments for subsequent flow cytometric characterization.

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 ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore essential for understanding pathogenicity of different pathogens including HIV. Gamma delta (GD) T cells are the prototype of &#39;unconventional&#39; T cells and represent a relatively small subset of T cells defined by their expression of heterodimeric T-cell receptors (TCRs) composed of gamma and delta chains. This sets them apart from the classical and much better known CD4+ helper T cells and CD8+ cytotoxic T cells that are defined by alpha-beta TCRs. GD T cells often show tissue-specific localization and are enriched in epithelium. GD T cells orchestrate immune responses in inflammation, tumor surveillance, infectious disease, and autoimmunity.
Here, we present a method to reproducibly isolate and analyze human endocervical intraepithelial GD T lymphocytes. We have used endocervical cytobrush samples from women participating in the Women&#39;s Interagency HIV Infection Study (WIHS). Knowledge about GD T cells interactions during conditions in which there is an insult to the vaginal mucosal could be applied to any clinical study in which mucosal vulnerability is addressed, including the development of vaginal microbicides.In addition, knowledge about mucosal GD T cell responses has potential for application of GD T cell-based immune therapy in treating infectious diseases.

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore ...

Isolation and Identification of Extravascular Immune Cells of the Heart

The immune system is an essential component of a healthy heart. The myocardium is home to a rich population of different immune cell subsets with functional compartmentalization both during steady state and during different forms of inflammation. Until recently, the study of immune cells in the heart required the use of microscopy or poorly developed digestion protocols, which provided enough sensitivity during severe inflammation but were unable to confidently identify small &#8212; but key &#8212; populations of cells during steady state. Here, we discuss a simple method combining enzymatic (collagenase, hyaluronidase and DNAse) and mechanical digestion of murine hearts preceded by intravascular administration of fluorescently-labelled antibodies to differentiate small but unavoidable intravascular cell contaminants. This method generates a suspension of isolated viable cells that can be analyzed by flow cytometry for identification, phenotyping and quantification, or further purified with fluorescence-activated cell sorting or magnetic bead separation for transcriptional analysis or in vitro studies. We include an example of a step-by-step flow cytometric analysis to differentiate the key macrophage and dendritic cell populations of the heart. For a medium sized experiment (10 hearts) the completion of the procedure requires 2&#8211;3 h.

This protocol presents a simple and efficient method to isolate, identify and quantify immune cells residing in the myocardium of mice during steady state or inflammation. The protocol combines enzymatic and mechanical digestion for the generation of a single cell suspension that can be further analyzed by flow cytometry.