This video demonstrates the immunolabeling of macrophage extracellular traps, METs, in bronchoalveolar macrophages from bronchoalveolar lavage fluid, and their visualization using a confocal microscope.
Protocol
All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.
1. Bronchoalveolar Lavage (BAL) Macrophages
Use BAL to obtain lung macrophages in (1) human subjects by bronchoscopy, and (2) mice by using intra-tracheal aspiration. 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 macrophage extracellular traps, or METs (this will need to be determined for each individual project).
Take the BAL solution and spin it 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.
If necessary, request a formal differential count; most of the cells (generally >80%) will be macrophages. NOTE: This will generally be performed by a clinical histology service using the morphologic appearance of the different cell types.
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.
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 to coverslips as mentioned above.
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.
Wash cells briefly in PBS, and then permeabilize in 0.2% Tween 20 in PBS for 20 min.
Block cells with 10% chicken sera in 5% bovine serum albumin (BSA) diluted in PBS for 30 min.
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.
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.
Wash the sections in PBS and mount with a 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.
Disclosures
The authors have nothing to disclose.
Materials
Human samples: Primary antibodies
Rabbit anti-human H3Cit (Citrulline R26)
Abcam
AB5103
10 ug/ml
Mouse anti-human MMP9
Novus Biological
AB119906
1:200 ascites, no concentration given
Rabbit anti-human PADI2/PAD2
Abcam
AB16478
7 ug/ml
Mouse anti-human PADI4/PAD4
Abcam
AB128086
10.1 ug/ml
Secondary antibodies
Chicken anti-rabbit AF 488/
Life technologies
A-21441
Chicken anti-rabbit AF 594
Life technologies
A-21442
Chicken anti-goat AF 594
Life technologies
a-21468
Chicken anti-mouse AF488
Life technologies
A-21200
Software Programs
Imaris
Bitplane
Image J
NIH
To average intensity of fluorphores a standard office application like Microsoft Excel can be used
Microscopes
C1 Confocal scanning microscope
Nikon
FV1200 Confocal scanning microscope
Olympus
Human BAL samples from the bronchoscopy suite at Monash Medical Centre