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…
開示
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 |
