In Vitro Stimulation and Visualization of Macrophage Extracellular Traps

Published: November 30, 2023

Abstract

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.

Protocol

1. Polarization of HMDM Under sterile conditions, prepare the M1 priming media by adding interferon γ (IFNγ; 20 ng/mL) and lipopolysaccharide (LPS; 1 μg/mL) to the complete RPMI-1640 culture media. Prepare the M2 priming media by adding interleukin 4 (IL-4; 20 ng/mL) to the complete RPMI-1640 culture media. Under sterile conditions, aspirate media from the tissue culture plate wells that contain the Human monocyte-derived macrophages, HMDM, which have been seeded and …

Açıklamalar

The authors have nothing to disclose.

Materials

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

Etiketler

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Bu Makaleden Alıntı Yapın
In Vitro Stimulation and Visualization of Macrophage Extracellular Traps. J. Vis. Exp. (Pending Publication), e21788, doi: (2023).

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