Induction and Visualization of Neutrophil Extracellular Traps in Pre-Labeled Neutrophils

Abstract

Source: Arends, E. J. et al., A High-throughput Assay to Assess and Quantify Neutrophil Extracellular Trap Formation. J. Vis. Exp. (2019)

This video demonstrates the induction and visualization of neutrophil extracellular traps or NETs in pre-labeled neutrophils using patient serum containing anti-neutrophil cytoplasmic antibodies. Furthermore, using an impermeable DNA dye and fluorescence microscopy enables the visualization of NETs.

Protocol

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.

1. Isolation of Healthy Neutrophils

1. Obtain 20 mL of peripheral blood from a healthy donor in two 10 mL ethylenediaminetetraacetic acid (EDTA)-coated tubes.
2. Put 10 mL of blood in a sterile 50 mL tube and add phosphate-buffered saline (PBS) up to 32.5 mL.
3. Add density gradient (e.g., Ficoll-amidotrizoaat) under the cells.
   1. Take up 14 mL of density gradient ....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Aqua Sterile H2O	B. Braun, Melsungen, Germany	12604052
Fetal bovine serum (FBS)	Bodinco, Alkmaar, The Netherlands		Used in high concentrations it could influcence NET formation
Ficoll 5,7% - amidotrizoaat 9% density 1,077 g / mL	LUMC, Leiden, The Netherlands	97902861
Immunofluorescence confocal microscope	Image Xpress Micro Confocal (Molecular Devices, Sunnyvale, CA, USA)
Neutralization PBS (10x)	Gibco, Paisley, UK	70011-036
Penicillin / streptomycin (p/s)	Gibco, Paisley, UK	15070063
Phenol red-free RPMI 1640 (1x)	Gibco, Paisley, UK	11835-063	Phenol red can interfere with the immunofluorescence signal
Phosphate-buffered saline (PBS)	B. Braun, Melsungen, Germany	174628062
PKH26 2 uM Red fluorescent cell linker	Sigma Aldrich Saint-Louis, MO, USA	PKH26GL-1KT	PKH are patented fluorescent dyes and a cell labeling technology named after their discoverer Paul Karl Horan
Program for scientific multidimensional images analysis	ImageJ, Research Services Branch, National Institute of Mental Health, Bethesda, Maryland, USA
RPMI medium 1640 (1x)	Gibco, Paisley, UK	52400-025
Sytox green 5 mM Impermeable DNA dye	Gibco, Paisley, UK	57020
Trypan blue stain 0,4%	Sigma Aldrich, Germany	17942E
96-well, black, flat-bottom, tissue culture-treated plate	Falcon, NY, USA	353219