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Dans cet article

  • Overview
  • Protocole
  • Déclarations de divulgation
  • matériels
  • Références

Overview

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.

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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Déclarations de divulgation

No conflicts of interest declared.

matériels

NameCompanyCatalog NumberComments
120Q broad spectrum fluorescent light sourceEXFO Photonic Solutions, Toronto, Canadax-cite series
Corning CellBIND Multiple Well Plate (12 wells)Sigma-AldrichCLS3336For cell culture
Hypochlorous acid (HOCl)Sigma-Aldrich320331For MET stimulation
Interferon gammaThermo-FisherPMC4031For M1 priming
Interleukin 4Integrated Sciencesrhil-4For M2 priming
Interleukin 8Miltenyl Biotec130-093-943For MET stimulation
L-GlutamineSigma-Aldrich59202CAdded to culture media
Olympus IX71 inverted microscopeOlympus, Tokyo, Japan
Phorbol 12- myristate 13-acetate (PMA)Sigma-AldrichP8139For MET stimulation
Phosphate buffered saline (PBS)Sigma-AldrichD5652For washing steps
RPMI-1640 mediaSigma-AldrichR8758For cell culture
SYTOX greenLife TechnologiesS7020For MET visulaization
TH4-200 brightfield light sourceOlympus, Tokyo, Japanx-cite series
Tumor necrosis factor alphaLonza300-01A-50For MET stimulation

Références

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