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

Published: October 31, 2023

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 Obtain 20 mL of peripheral blood from a healthy donor in two 10 mL ethylenediaminetetraacetic acid (EDTA)-coated tubes. Put 10 mL of blood in a sterile 50 mL tube and add phosphate-buffered saline (PB…

Disclosures

The authors have nothing to disclose.

Materials

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

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Cite This Article
Induction and Visualization of Neutrophil Extracellular Traps in Pre-Labeled Neutrophils. J. Vis. Exp. (Pending Publication), e21656, doi: (2023).

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