Source: Zhang, Y., et al. In Vitro Stimulation and Visualization of Extracellular Trap Release in Differentiated Human Monocyte-derived Macrophages. J. Vis. Exp. (2019).
This video demonstrates in vitro stimulation of macrophage extracellular traps (METs) in human monocyte-derive macrophages using different inflammatory stimuli such as TNF-alpha, PMA, and HOCL. The video also demonstrates the method of SYTOX dye staining of METs for visualization.
1. Polarization of HMDM
2. Stimulation of HMDM to induce MET Release
3. Visualization of MET in Live Cell Culture
The authors have nothing to disclose.
120Q broad spectrum fluorescent light source | EXFO Photonic Solutions, Toronto, Canada | x-cite series | |
Corning CellBIND Multiple Well Plate (12 wells) | Sigma-Aldrich | CLS3336 | For cell culture |
Hypochlorous acid (HOCl) | Sigma-Aldrich | 320331 | For MET stimulation |
Interferon gamma | Thermo-Fisher | PMC4031 | For M1 priming |
Interleukin 4 | Integrated Sciences | rhil-4 | For M2 priming |
Interleukin 8 | Miltenyl Biotec | 130-093-943 | For MET stimulation |
L-Glutamine | Sigma-Aldrich | 59202C | Added to culture media |
Olympus IX71 inverted microscope | Olympus, Tokyo, Japan | ||
Phorbol 12- myristate 13-acetate (PMA) | Sigma-Aldrich | P8139 | For MET stimulation |
Phosphate buffered saline (PBS) | Sigma-Aldrich | D5652 | For washing steps |
RPMI-1640 media | Sigma-Aldrich | R8758 | For cell culture |
SYTOX green | Life Technologies | S7020 | For MET visulaization |
TH4-200 brightfield light source | Olympus, Tokyo, Japan | x-cite series | |
Tumor necrosis factor alpha | Lonza | 300-01A-50 | For MET stimulation |