Visualization of Extracellular Traps in Macrophages from Human Bronchoalveolar Lavage Fluids
Published: October 31, 2023
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 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 bron…
Divulgazioni
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 | |||
| 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 |
Tags
Citazione di questo articolo
Visualization of Extracellular Traps in Macrophages from Human Bronchoalveolar Lavage Fluids. J. Vis. Exp. (Pending Publication), e21722, doi: (2023).