Visualization of Extracellular Traps in Macrophages from Human Bronchoalveolar Lavage Fluids

Abstract

Source: Sharma, R. et al., Visualizing Macrophage Extracellular Traps Using Confocal Microscopy. J. Vis. Exp. (2017)

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

1. 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).
   1. 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.
   2. 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.
   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 to 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 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.

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
Media
RPMI	Sigma-Aldrich	r8758
Fetal calf serum	Sigma-Aldrich	F0926
L-glutamine	Sigma-Aldrich	G7513
Other reagents
paraformaldehyde/periodate/lysine (PLP) fixative	Sigma-Aldrich	27387
Natural formalin	Sigma-Aldrich	42904
Coverslips 12 ml	Sigma-Aldrich	S1815
Coverslips 60 x 24 ml	Sigma-Aldrich	C6875