In Vitro Stimulation and Visualization of Macrophage Extracellular Traps

Protocole

1. Polarization of HMDM

1. 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.
2. Under sterile conditions, aspirate media from the tissue culture plate wells that contain the Human monocyte-derived macrophages, HMDM, which have been seeded and cultured as described in section 1.
3. Carefully wash the wells containing the cells 3x with sterile PBS (pre-warmed to 37 °C), using 1 mL....

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matériels

Name	Company	Catalog Number	Comments
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