eoe sub articles

EoE Sub Articles

1. Polarization of HMDM
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	<li>Under sterile conditions, prepare the M1 priming media by adding interferon &#947; (IFN&#947;; 20 ng/mL) and lipopolysaccharide (LPS; 1 &#956;g/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.</li>
	<li>Under sterile conditions, aspirate media from the tissue culture plate wells that contain the Human monocyte-derived macrophages, HMDM, which have been seeded and ...

in vitro stimulation and visualization of macrophage extracellular traps

Source: Zhang, Y., et al. <a href="https://app.jove.com/v/60541">In Vitro Stimulation and Visualization of Extracellular Trap Release in Differentiated Human Monocyte-derived Macrophages.</a> 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.

In Vitro Stimulation and Visualization of Macrophage Extracellular Traps

1. Polarization of HMDM

	Under sterile conditions, prepare the M1 priming media by adding interferon &#947; (IFN&#947;; 20 ng/mL) and lipopolysaccharide ...

Begin with an adherent macrophage culture.
Add pro-inflammatory cytokines.
The cytokine binds to the macrophage surface receptors, initiating a series of inflammatory events.
First, the cytokine signaling triggers the production of antimicrobial peptides.
Simultaneously, the chromatin within the macrophage nucleus undergoes decondensation.
This process, coupled with changes in membrane composition, disrupts the nuclear membrane, releasing decondensed chromatin into the cytoplasm.
In the cytoplasm, the antimicrobial peptide interacts with the released chromatin.
Cytokine signaling-mediated cell membrane permeabilization causes the decondensed chromatin to be released into the extracellular space, forming a Macrophage Extracellular Trap, or MET.
These web-like structures consist of DNA, histones, antimicrobial molecules, and enzymes.
Add SYTOX green, a fluorescent nucleic acid dye that selectively binds to the exposed DNA in the extracellular traps.
Under a fluorescence microscope, the MET appears as green streaks of extracellular DNA released from the stimulated macrophage.