An In Vitro Technique to Generate Human Neutrophil Extracellular Traps

Abstract

Source: Najmeh, S., et al. Simplified Human Neutrophil Extracellular Traps (NETs) Isolation and Handling. J. Vis. Exp. (2015)

This video demonstrates an in vitro technique to generate and isolate neutrophil extracellular traps (NETs). Neutrophils are isolated from peripheral blood and stimulated with phorbol myristate acetate (PMA) to produce NETs. During incubation, the neutrophils undergo a process of cell death (NETosis) to release NETs comprising decondensed chromatin mixed with granular cytoplasmic proteins.

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. NETs Generation

1. Stimulate neutrophils with 500 nM of PMA (per 30 ml of neutrophil solution) and incubate on a 150 x 25mm flat tissue culture dish with a 20 mm grid for 4 hr at 37 °C 5% CO₂. This will allow NETosis.
2. After 4 hr of stimulation, gently aspirate and discard the media, leaving the layer of NETs and neutrophils adhered at the bottom. Do not disrupt this l....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dulbecco's Phosphate-Buffered Saline (PBS), Modified, without calcium chloride and magnesium chloride	Wisent	311-425-CL
RPMI-1640 Medium with L-glutamine	Wisent	350-000-CL	3% RPMI solution is made by supplementing RPMI with 3% Fetal Bovine serum
Phorbol 12-myristate 13-acetate (PMA)	Sigma-Alrdrich	P8139
Fetal Bovine Serum (FBS)	Wisent	080-150
Integrid tissue culture dish with 20mm grid (150 x 25mm)	Falcon	353025