Overview
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.
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Materials
Name | Company | Catalog Number | Comments |
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 |