Visualizing Macrophage Extracellular Traps Using Confocal Microscopy

This protocol follows experiments approved in: (1) humans by the Ethics Committee of Monash Medical Centre, and (2) animals by the Ethics Committee of the University of Melbourne.

1. Bronchoalveolar Lavage (BAL) Macrophages

1. Use BAL to obtain lung macrophages in: (1) human subjects by bronchoscopy⁶, and (2) mice by using intra-tracheal aspiration¹³.
   NOTE: Acquisition of the macrophages generally causes some activation of these cells. Macrophages are obtained from patients who require a bronchoscopy to investigate a potential medical problem and not all subjects may be suitable for analysis of METs (this will need to be determined for each individual project).
   1. Take the BAL solution and spin down (500 x g for 10 min) to form a pellet, wash twice with culture medium (Roswell Park Memorial Institute Medium (RPMI) with 5% fetal calf serum and 1% L-glutamine) at room temperature, and then dilute cells in 4 mL of culture medium.
   2. If necessary, request a formal differential count; most of the cells (generally >80%) will be macrophages⁶.
      NOTE: This will generally be performed by a clinical histology service using the morphologic appearance of the different cells types⁶.
   3. Perform a viable macrophage cell count on the BAL solution using Trypan blue exclusion and a hemocytometer.
      NOTE: BAL solution is obtained from humans and mice as mentioned in step 1.1.
   4. Add 1 – 4 x 10⁵ macrophages to 24-well plates on coverslips in 500 µL of culture medium per well and leave overnight at 37°C; the cells will adhere to the coverslips. For human samples, add antibiotics (e.g., penicillin and streptomycin, 10⁴ U/mL) to prevent bacterial contamination.

2. Immunofluorescence Labeling/Microscopy of BAL Macrophages

NOTE: Cells are adhered onto coverslips as mentioned above.

1. Remove the culture medium and wash the cells once with phosphate buffered saline (PBS). Fix cells in 2% paraformaldehyde/periodate/lysine (PLP) fixative for 10 min.
2. Wash cells briefly in PBS, and then permeabilize in 0.2% Tween 20 in PBS for 20 min.
3. Block cells with 10% chicken sera in 5% bovine serum albumin (BSA) diluted in PBS for 30 min.
4. Incubate with primary and isotype control antibodies for 1 h at room temperature at 1:100 concentrations (more specific details are listed in Table of Materials) at 37 °C.
   NOTE: For the BAL samples, a specific marker of macrophages is usually not required.
5. After the addition of primary antibodies, wash the cells in PBS, and further incubate with the corresponding secondary antibodies for 40 min at room temperature.
6. Wash the sections in PBS and mount with DAPI-based mounting medium for visualization using a confocal microscope (as mentioned above) at laser excitations 405, 488, 561 and 647 nm and a 40X, 1.0 NA oil objective.

3. Lung Tissue Samples

NOTE: For in vivo studies of lung tissue, study the samples after the relevant exposure (e.g., as described by O'Sullivan et al.⁷). The exposure that is most relevant for this experiment are infectious microorganisms, particularly bacteria. Mouse strains that could be used for this method are C57BL/6 or BALB/c mice, 10 – 12 weeks old, weight 25 – 30 g, and male.

1. Euthanize mice, via an intraperitoneal injection of ketamine (400 mg/kg) and xylazine (40 mg/kg). Open the thoracic cavity and expose the lungs and heart using surgical forceps and scissors.
   1. Infuse warm PBS using a 21-gauge needle into the right ventricle to wash out the blood from the vessels for 30 s, then infuse 10 mL of 2% PLP fixative for 30 s (into the right ventricle).
   2. Use warm PBS (42 °C) to lavage the open chest cavity. Intubate the trachea with a 21-gauge needle and a piece of 0.8 mm tubing cut to size, and inject 800 µL of pre-warmed (to 42 °C), low-melting point 3% agarose solution.
   3. Tie off the trachea with braided silk (4.0 USP). Place the thread around the trachea, just below the insertion point of the cannula, and secure it via a simple overhand knot. To solidify the agarose, administer ice-cold PBS around the lungs. Remove the lungs and heart as a block from the thoracic cavity using both scissors and blunt dissection. Remove each lung individually and then fix them for 16 h in 2% PLP or formalin at 4 °C.
   4. Store the specimens in PBS at 4 °C until tissue sectioning. These methods for obtaining lung tissue have been previously described¹³.
2. To prepare the lung tissue samples use the following steps.
   1. Fix lung tissues (resected in step 3.1) tissue in 10% natural-buffered formalin for 24 h at room temperature. Transfer to 75% ethanol and put in a tissue processor on a 12 h cycle, (2.5 h in 75% EtOH, 3 h in absolute EtOH, 3.5 h in solvent 3B, and for 3 h in paraffin).
   2. Embed in standard paraffin to create formalin-fixed, paraffin-embedded (FFPE) blocks which are stored at room temperature.
      NOTE: At this stage, it is generally convenient to cut the lung sections for standard slides.
   3. Cut the FFPE lung sections at 4 – 5 µm thickness using a microtome and mount on charged, coated slides as previously described by O'Sullivan et al.⁷
   4. To mount slides, put 3 drops of mounting medium on 60 mm x 24 mm coverslips (size 1.5), invert the slide on the coverslip and push out excess medium. Hermetically seal with nail polish and store at room temperature.

4. Preparation/Microscopy of Lung Tissue Samples

1. To de-wax, rehydrate and pretreat the FFPE lung tissue sample with antigen retrieval solution.
   1. Oven dry the FFPE slides for 60 min at 60 °C. Then put the slides in xylene solution for 30 min and transfer to 70% ethanol solution for 5 min at room temperature. Rinse in tap water.
   2. Place in heat-proof plastic wrap and subject to HIER-antigen retrieval in a pressure cooker for 10 min in 10 m/mol/L Tris, 1 mmol/EDTA pH 9.0. Cool for 20 min. Wash twice in tap water for 5 min on a rocker, then once with PBS on rocker.
   3. Block with 10% chicken serum in 5% BSA/PBS for 30 min at room temperature.
2. Stain the tissue.
   1. To define METs in macrophages, add primary antibodies (MMP-9, H3Cit, and F4/80) in 1% BSA/PBS for 16 h at 4 °C at 1/100 dilution (specific details about antibody amounts are listed in Table of Materials). Wash two times with PBS for 5 min on a rocker.
   2. Achieve fluorescent detection by incubation with corresponding secondary antibodies in 1% BSA/PBS for 40 min at room temperature. Antibodies are: (1) chicken anti-mouse 488 (green), (2) chicken anti-goat 594 (red), and (3) donkey anti-mouse 647 (far red).
   3. Wash the sections in PBS and mount with DAPI-containing mounting medium for chromatin staining.
3. Obtain fluorescent images using a confocal laser scanning head attached to an inverted microscope.
   1. Excite the preparation with 405, 488, 561, and 647 nm lasers. Capture single plane 512 x 512-pixel images by clicking on the line-sequential, leveling button (2 line averaging) using 20X 0.1 NA air and 40X 1.0 NA oil objectives.
   2. Obtain at least ten fields of view per section for analysis and data for each result (i.e., 10 high-power fields of view (HFOV) for the control, 10 for the stimulated sample, etc., for each mouse).
      NOTE: To obtain a representative sample of the whole field, a matrix system can be used in which the field is divided into different sections and for each different sample the same matrix is used to select fields of view (e.g., the field can be divided into 9 sections, and from the center and the 4 corners, two HFOV are taken).

5. Three-dimensional (3-D) Imaging of METs

NOTE: As METs are 3-D structures, by taking multiple z-stack images, which are reconstituted, may give valuable information.

1. Section the lung specimens from paraffin blocks, at 200 µm using a vibratome at an amplitude set at 1.8 mm and speed of 0.1-0.5 mm/s.
2. Block lung tissue with 20% of the respective serum (10% fetal calf serum and 10% chicken serum) and then permeabilize in PBS supplemented and 3.75% Triton-X 100 for 7 h at room temperature under gentle agitation.
3. Stain the sections for 16 h at 4 °C with the relevant primary antibodies (MMP-9, H3Cit, and F4/80) in 200 µL volumes in microcentrifuge tubes (antibody concentrations are listed in Table of Materials).
4. Wash the sections in PBS, 3 times for 10 min before incubating with relevant secondary antibodies at room temperature for 1 h.
5. Stain the lung sections for DAPI (1:5,000) in solution for 20 min, before adding the cover slips to the microscope slides in PBS.
6. Obtain fluorescence images using an inverted confocal microscope (40x 1.3 NA) equipped with 405 nm, 440 nm, 473 nm, 543 nm, and 635 nm lasers.
7. Record 3-D z-stacks of 0.54 µm thickness with optimal z-sectioning for the 40x 1.3 NA oil objective.
   NOTE: The software used will vary depending on the individual microscope. An example of how the software can be used for one microscope is provided in Supplementary File 1.

6. Image Analysis

NOTE: The analysis of samples requires the use of specific imaging analysis software and examples are listed in Table of Materials. While the analysis of results will depend on the specific program used, the listed points below are important.

1. Analysis of BAL samples
   1. Define the number of macrophages by selecting the blue channel for DAPI and assigning a minimum size for the cells (e.g., > 18 µm in diameter). Using the relevant software, measure the total number of macrophages per field.
   2. Count the number of cells that have the features of a MET by the presence of extracellular chromatin detected by DAPI with co-expression of other markers (e.g., MMP12, PAD2, and H3Cit).
      NOTE: The number of markers that can be analyzed depends on the number of lasers available, but ideally in addition to DAPI, at least two other markers should be included. Other less specific parameters for MET expression (e.g., number of cells with extracellular chromatin and an enlarged nucleus) can be used.
   3. To compare the MET expression in BAL samples (between the control, smoke, and smoke/DNase treated groups), analyze a minimum of 100 BAL macrophages per sample.
   4. To measure secondary antibody and isotype control levels of fluorescence; measure a minimum of 100 cells in each sample for their fluorescence. Measure the average level of background fluorescence for each marker (e.g., H3Cit) using standard software.
   5. To define MET expression, count the cells with the typical phenotype (e.g., extracellular chromatin with co-expression of H3Cit and MMP9) and for each MET, measure the level of fluorescence of markers (e.g., H3Cit and MMP9). Exclude cells producing METs if their staining was not above background and isotype control.
   6. For human BAL samples, analyze a minimum of 100 cells for each sample for the percentage of macrophages that make METs. For murine samples, as the cells are smaller and METs are harder to define, analyze a larger number of cells for each sample (e.g., 200 macrophages).
2. Analysis of lung tissue samples
   1. To determine the presence of METs in lung tissue, use specific macrophage marker such as F4/80.
   2. Define the presence of METs in lung tissue by using the co-localization of F4/80 in cells that express extracellular chromatin with other parameters as listed in section 6.1.
   3. Determine the levels of background fluorescence in samples with secondary antibody and the isotype controls. Exclude METs from analysis that are not above background.