Isolation and analysis of macrophage subsets from the mouse and human lung

Abstract

Pulmonary macrophages are heterogeneous. Distinct populations of resident tissue macrophages exist in the lung airspace and tissue compartments during homeostasis. During inflammation, these are joined by monocyte-derived recruited macrophages. Flow cytometry can be used to identify and purify lung macrophage subsets. Here we describe methods for identifying and isolating macrophages from bronchoalveolar lavage and digested lung tissues from mouse and human. We also describe basic staining for flow cytometry analysis of different macrophage subsets.

1. Introduction

In the lung, macrophages exist in two distinct compartments. Airspace macrophages (AM) reside in airspaces lumen, whereas interstitial macrophages (IM) reside in the tissues. Macrophage gene expression and function are influenced by factors in the local environment [1]. Therefore, the ability to distinguish macrophages from the airspaces versus interstitial compartments of the lung is essential for studies of macrophage function in health and disease. Isolation of IMs requires digestion of the lung tissue, and different digestion methods will alter the yield of macrophages. Digestion of the adult mouse lung with Liberase has been shown to provide the greatest yield of distinct macrophage subsets compared to collagenase or elastase [2]. Similarly, enzymatic digestion with Liberase has been shown to provide the most consistent results for studying IM subsets from human lung tissue [3]. Within the IM population, several subsets have been described [4,5]. These can be distinguished using cell surface markers. Surface markers can be used to distinguish AMs from IMs during homeostasis.

During inflammation circulating monocytes traffic to the lung and differentiate into macrophages in both the airspace and interstitium. These recruited macrophages are distinct from tissue resident macrophages and play distinct roles during inflammation and tissue repair. Lung resident macrophages are thought to suppress inflammation and promote resolution and tissue homeostasis, whereas recruited macrophages are shown to have gene expression consistent with promoting inflammation [6–8]. Unfortunately, surface markers have not been identified to distinguish recruited macrophages in the airspace from recruited macrophages in the tissue compartment. To confidently separate recruited macrophages by compartment, anti-CD45 antibody can be given to mice via intratracheal instillation after euthanasia but prior to lavage and lung digestion, labeling all airspace immune cells [9]. This technique can be adapted to human tissue if there is access to a whole lobe.

Identifying markers that are both suitable for flow cytometry and allow discrimination of macrophage subsets is challenging given the heterogeneity and phenotypic plasticity of macrophages in the lung. Additionally, other mononuclear phagocytes such as dendritic cells and monocytes are found in the lung. To distinguish macrophages from these other mononuclear phagocyte populations in the mouse lung, the pan-macrophage markers CD64 and MertK can be used [10,11]. CD88, which is also highly expressed on lung macrophage [12] can further enhance discrimination. In the human lung, CD206 distinguishes macrophages from other mononuclear phagocyte populations [13].

In this chapter, we describe methods to isolate pulmonary macrophages from the airspace and interstitium in mouse and human lungs. We also provide flow cytometry methods for identification and analysis of different macrophage populations in both mice and humans.

2. Materials

2.1. Labeling of mouse airspace cells with anti-CD45

1. Fluorochrome-conjugated rat anti-mouse CD45 antibody (clone 30-F11)

2. 1X PBS without calcium or magnesium

3. 1 mL syringe

4. Scissors

5. Forceps

6. 18-gauge IV catheter

2.2. Single-cell suspensions of mouse BAL and lung tissue

1. Syringes: 1 mL, 10 mL

2. 25-gauge needles

3. 35 mm × 10 mm round culture dishes or 12-well tissue culture plate

4. Pentobarbital sodium

5. 1X PBS

6. PBS-EDTA: 1X PBS with 5 mM EDTA

7. Hank’s Buffered Salt Solution (HBSS)

8. FACS buffer: 1X HBSS with 0.06% BSA, 0.3 mM EDTA

9. Liberase TM (Roche), 2 mg/mL stock solution

10. 100 mM EDTA

11. Razor blade

12. 100 μm and 70 μm nylon filters

13. 37°C incubator with shaker

14. Glass pipet with a bulb

2.3. Staining mouse lung samples for flow cytometry

1. FACS buffer

2. FACS tubes or v-bottom 96-well plates

3. Fluorochrome-conjugated antibodies (see Table 1)

4. Rat anti-mouse CD16/CD32 (Fc receptor block, clone 93)

5. UltraComp eBeads (Invitrogen) or comparable compensation beads (see Note 1)

6. Centrifuge

7. Flow cytometer

Table 1.

Antibodies and dyes for identifying macrophages in mouse BAL and digest

Marker	Clone	Suggested fluorochromes	Working concentration (μg/mL)	Host species	Target species
Live/Dead dye (e.g. DAPI)			1 μg/mL
CD45	30-F11	BUV395	0.5 μg/mL	Rat	Mouse
Ly6G	1A8	BV421	1.25 μg/mL	Rat	Mouse
CD64	X54–5/7.1	PE-Cy7	0.5 μg/mL	Mouse	Mouse
MerTK	Polyclonal	Biotinylated	2 μg/mL	Goat	Mouse
CD88	20/70	PerCP-Cy5.5	0.5 μg/mL	Rat	Mouse
SiglecF	E50–2440	APC	0.5 μg/mL	Rat	Mouse
CD11b	M1/70	BUV737	0.5 μg/mL	Rat	Mouse
CD11c	N418	APC-Cy7	0.5 μg/mL	Hamster	Mouse
CD206	C068C2	FITC	1.25 μg/mL	Rat	Mouse

2.4. Single-cell suspension of human lung tissue

1. Scissors

2. Razor blade

3. gentleMACS Dissociator (see Note 3)

4. gentleMACS C tubes

5. 37°C incubator

6. Sterile gauze

7. 70 μm nylon filters

8. Digestion buffer: RPMI with 0.2 mg/mL Collagenase D (Sigma-Aldrich), 0.2 mg/mL DNAse I (Sigma-Aldrich)

9. Hank’s Buffered Salt Solution (HBSS)

10. FACS buffer: 1X PBS with 10% BSA

2.5. Staining human lung samples for flow cytometry

1. FACS buffer

2. FACS tubes

3. Fluorochrome-conjugated antibodies (see Table 2)

4. Anti-human CD16/CD32 (Fc receptor block, polyclonal)

5. Centrifuge

6. Flow cytometer

Table 2.

Antibodies and dyes used for identifying macrophages in human lung digest (see Note 2)

Marker	Clone	Suggested fluorochromes	Working concentration (μg/mL)	Host species	Target species
Live/Dead dye (e.g. DAPI)			1 μg/mL
CD206	15–2	PE-Cy7	0.12 μg/mL	Mouse	Human
CD43	84–3C1	APC	0.5 μg/mL	Mouse	Human
CD45	HI30	BUV395	1 μL/test	Mouse	Human

3. Methods

3.1. Labeling of mouse airspace cells with anti-CD45

1. Euthanize mouse with pentobarbital sodium by intraperitoneal injection (see Note 4).

2. Place mouse supine on a board and immobilize the head by placing a rubber band around the board and hooking it under the teeth. Pin the tail and arms to the board (do not over stretch the arms away from the body).

3. Spray fur over the neck with 70% ethanol. Lift neck skin using forceps and make a small incision. Use scissors and forceps to gently resect the skin of the neck. The submandibular glands are easily visible. Gently pull apart with forceps to expose the trachea. Use forceps to carefully remove the fascia and small muscles overlying the trachea.

4. With the bevel facing upward, insert the catheter with the needle into the trachea in between the largest and most proximal cartilage rings. Slide the plastic cannula down the trachea and remove the needle. The cannula should be secure in the trachea but not inserted so deeply that it enters the right or left lung.

5. Attach a 1 mL syringe containing 800 μL of fluorochrome-conjugated anti-CD45 antibody diluted 1:100 in PBS (final concentration 2 μg/mL). Instill the anti-CD45 antibody and leave in the lung for 4 minutes. Withdraw fluid and collect in a 15 mL conical tube or FACS tube kept on ice.

3.2. Single-cell suspension of mouse airspace cells from bronchoalveolar lavage (BAL)

1. Lavage with 1 mL of room temperature PBS-EDTA four times and collect in the same tube. The final BAL volume is approximately 4.8 mL (see Note 5).

2. Centrifuge the BAL fluid at 300×g for 5 min at 4°C.

3. Remove the supernatant by aspiration or decanting, and resuspend the cell pellet in the FACS buffer or directly in the Fc blocking buffer (see section 3.4) for staining.

3.3. Single-cell suspension of mouse lung tissue

1. Cut skin and parietal peritoneal membrane circumferentially below the ribs. Gently retract the abdominal organs to visualize the diaphragm.

2. Use scissors to create a small incision in the diaphragm directly below the heart, taking care not to puncture the lungs. Once the lungs have collapsed, carefully cut away the diaphragm to expose the thoracic cavity. Cut through the sternum from caudal to rostral. Separate the rib cage laterally to expose the lungs. Pin the rib cage to the board.

3. Cut aorta near the kidneys to allow blood to drain. Insert a 25g needle with a 10 mL syringe of cold PBS into the right ventricle. Perfuse the lungs until they are white.

4. Dissect out lungs, including the heart and thymus, starting at the proximal trachea. Place the lungs in the culture dish or 12-well plate on ice.

5. Remove each lobe of the lungs. Mince lung into very fine pieces with a razor blade and pour minced lung into pre-chilled plate on ice.

6. Dilute Liberase 1:5 in HBSS and add 1 mL to each well of the plate (this amount is for the whole lung).

7. Incubate for 25 min at 37°C on a shaker. After incubation, add 50 μL of 100 mM EDTA to each well to inhibit further digestion. Use a glass pipet with a bulb to further dissociate the tissue until single cell suspension is achieved. Leave on ice until all samples are done.

8. Pre-wet 100 and 70 μm filters with HBSS. Pipet the cell suspension through the 100 μm filter and collect in a 50 mL conical tube. Rinse the well with 1–2 mL of HBSS and add to the filter.

9. Pellet cells at 300×g for 5 min, discard supernatant and resuspend pellet 1 mL of HBSS.

10. Pipet filtrate through the 70 μm filter and collect in another 50 mL tube. Rinse the first tube with 20 mL of HBSS and pour through the same 70 μm filter. Repeat for all samples.

11. Centrifuge at 300×g for 5 min at 4°C.

12. Aspirate or decant the supernatant. Resuspend the cell pellet in 1 mL of FACS buffer.

13. Load 100 μl (1/10^th) of each digest sample into wells of v-bottom 96-well plate or individual FACS tubes for staining. Keep on ice. Use leftover digest sample for an unstained sample and for single stain compensation controls if desired (see Note 1).

3.4. Staining mouse lung samples for flow cytometry

1. Prepare Fc blocking buffer for staining by diluting unconjugated anti-CD16/anti-CD32 antibody cocktail 1:100 in FACS buffer (final concentration 5 μg/mL), enough for 100 μL per stained sample, plus 100 μL for controls.

2. Prepare a 2X antibody master mix in FACS buffer. We find that a final 1:400 dilution for most antibodies works well, meaning the 2X cocktail has 1:200 dilutions (see Table 1). Make enough for 100 μL per stained sample.

3. Pellet cells at 300×g for 5 min at 4°C.

4. Resuspend cell pellets in 100 μL of Fc blocking buffer and incubate at 4°C or on ice for 5 minutes.

5. Add 100 μL of 2X staining cocktail over top, vortex or pipet to mix, and incubate at 4°C or on ice, covered to protect from light, a further 30–60 minutes (see Note 6). Add individual antibodies to single stain tubes or wells.

6. Washing in 96-well plate: Pellet cells at 300×g for 2 min at 4°C. Discard supernatant and blot plate. Resuspend cells in 200 μL of the FACS buffer and pellet again at 300×g for 2 min at 4°C. Discard supernatant again and blot plate. Resuspend cells a final time in 200 μL of the FACS buffer and pellet at 300×g for 2 min at 4°C. Discard supernatant.

   Washing in flow tubes: Wash cells by adding 2 mL of the FACS buffer to each sample and centrifuging at 300×g for 5 min. Discard supernatant.

7. Resuspend in 500 μL of the FACS buffer and analyze.

3.5. Tissue digestion for isolation of macrophages from human lung tissue

1. Cut a 15 cm³ cube of lung tissue (2.5 cm per side) from the distal part of the lobe, avoiding large airways, vessels or pleural surface (unless those are desired for study).

2. Mince lung tissue into small pieces using scissors.

3. Further chop with a razor blade into as fine as specimen as possible and place tissue into a gentleMACS C tube.

4. Add digestion buffer to the tube to bring the final volume, including tissue, to 10 mL. Approximately 8 mL of digest buffer is needed. Load into the gentleMACS.

5. Run manufacturer’s program m_lung_01_02 (standard) 5 times.

6. Incubate sample for 30 min at 37°C under continuous rotation.

7. Place tube back into gentleMACS and run the manufacturer’s program m_lung_01_02 (standard) again 5 times.

8. Load the sample into 50 mL conical tube and add chilled HBSS to fill to 50 mL volume, mix well.

9. Centrifuge for 5 min at 300×g and carefully aspirate supernatant.

10. Add another 45 mL chilled HBSS and mix well, centrifuge for 5 min at 300×g, and again carefully remove the supernatant.

11. Resuspend the cell pellet in 5 mL chilled HBSS.

12. Pre-wet a 70 μm nylon filter with HBSS, then pass the sample through the filter. Centrifuge the sample for 5 min at 300×g with the filter in place. Gently aspirate the supernatant.

13. Resuspend the cells in 1 mL of sterile MACS Buffer (see Note 7).

3.6. Staining human lung samples for flow cytometry

1. Prepare Fc blocking buffer for staining by diluting unconjugated anti-CD16/anti-CD32 antibody cocktail 1:100 in FACS buffer (final concentration 10 μg/mL), enough for 100 μL per stained sample, plus 100 μL for controls.

2. Prepare a 2X antibody master mix in FACS buffer. A final 1:100 dilution for most antibodies works well, meaning the 2X cocktail has 1:50 dilutions (see Table 2). Make enough for 100 μL per stained sample.

3. Pellet cells at 300×g for 5 min at 4°C. Discard supernatant.

4. Resuspend cell pellets in 100 μL of Fc blocking buffer and incubate at 4°C or on ice for 5 minutes.

5. Add 100 μL of 2X staining cocktail over top, vortex to mix, and incubate at 4°C or on ice, covered to protect from light, 30–60 minutes.

6. Add 2 mL of the FACS buffer per tube to wash, pellet cells at 300×g for 5 min. Discard supernatant and repeat with 1 mL of the FACS buffer for a second wash.

7. Resuspend pellet in 500 μL of the FACS buffer. Add live/dead stain (e.g. DAPI, final concentration 1 μg/mL) and analyze.

4. Notes

1. Compensation controls should include single stains of each antibody used as well as an unstained cell sample. Although excess lung digest cells may be used for single stains, compensation beads are recommended because the digests contain heterogeneous cell populations with varying amounts of auto-fluorescence.

2. The antibodies listed in Table 2 allow identification of resident airspace macrophages and resident interstitial macrophages in a non-inflamed human lung. Markers for identifying recruited macrophages in the airspace and interstitium in the human lung in the setting of inflammation are not well defined.

3. This protocol uses a gentleMACS dissociator for mechanical dissociation. If this instrument is not available to you, the sample can be placed in a 50 mL conical tube with digestion buffer and steps 5–7 can be replaced with a 30 min incubation at 37°C with vigorous shaking.

4. We prefer to euthanize animals with pentobarbital sodium (Fatal-Plus) over CO₂ inhalation since the latter can affect macrophages. Cervical dislocation should be avoided because it can damage the trachea and cause bleeding in the lungs.

5. Do not lavage with more than 1 mL of PBS at a time. Excess fluid in the lungs can cause bleeding, and may lead to rupture and loss of BAL fluid into the pleural space.

6. Most antibodies will bind sufficiently in 30 minutes; however, MerTK and CD64 antibodies often require longer incubation.

7. Some investigators include an enrichment step at this point. Positive selection enrichment with CD45 microbeads using Miltenyi Biotec magnetic separation protocols can expedite sample acquisition times on the flow cytometer.

Figure 1.

Identification of macrophages from digested mouse lung. (a) Gating strategy to identify airspace macrophages and interstitial macrophages in the naïve mouse lung. Live cells are selected using side-scatter (SSC) and forward-scatter (FSC), and doublets are excluded with SSC-A and SSC-W. Neutrophils are excluded using Ly6G, and macrophages are identified by CD64 and CD88. Airspace macrophages (AMs) are separated from interstitial macrophages (IMs) by IT anti-CD45 antibody staining. AMs can then be separated into resident (ResAMs) and recruited (RecAMs) populations using SiglecF and CD11b. CD206 and CD11c staining can further subdivide IMs into IM 1/2 and IM 3 subsets. (b) Gating of AM and IM subsets in the inflamed lung. 6 days after intratracheal LPS administration, mice were euthanized, anti-CD45 antibody was instilled into the trachea, and lungs digested. AMs were distinguished from IMs using anti-CD45 antibody staining and resident versus recruited macrophage subsets identified.

Figure 2.

Identification of airspace macrophages from mouse bronchoalveolar lavage (BAL). (a) Live cells, doublet exclusion, and neutrophil exclusion are the same as Fig 1. AMs are separated into resident and recruited AMs by SiglecF and CD11b. (b) Gating strategy in BAL from the inflamed mouse lung. BAL was collected 6 days after intratracheal LPS treatment.

Figure 3.

Identification of AMs and IMs from non-inflamed human lung tissue. Live cells are selected using SSC and FSC, and doublets are excluded with FSC-A and FSC-H. Live cells are selected with a live/dead stain (e.g. DAPI), and leukocytes are identified with CD45. Macrophages are identified as AMs or IMs using CD206 and CD43.