Simplified Human Neutrophil Extracellular Traps (NETs) Isolation and Handling

Summary

In the following protocol, we describe a very simple way to isolate Neutrophil Extracellular traps (NETs) from human whole blood using readily available reagents. We then demonstrate how the isolated NETs can be used in an in vitro adhesion assay with cancer cells.

Abstract

Neutrophil Extracellular Traps (NETs) have been recently identified as part of the neutrophil’s antimicrobial armamentarium. Apart from their role in fighting infections, recent research has demonstrated that they may be involved in many other disease processes, including cancer progression. Isolating purified NETs is a crucial element to allow the study of these functions.

In this video, we demonstrate a simplified method of cell free NET isolation from human whole blood using readily available reagents. Isolated NETs can then be used for immunofluorescence staining, blotting or various functional assays. This enables an assessment of their biologic properties in the absence of the potential confounding effects of neutrophils themselves.

A density gradient separation technique is employed to isolate neutrophils from healthy donor whole blood. Isolated neutrophils are then stimulated by phorbol 12-myristate 13-acetate (PMA) to induce NETosis. Activated neutrophils are then discarded, and a cell-free NET stock is obtained.

We then demonstrate how isolated NETs can be used in an adhesion assay with A549 human lung cancer cells. The NET stock is used to coat the wells of a 96 well cell culture plate O/N, and after ensuring an adequate NET monolayer formation on the bottom of the wells, CFSE labeled A549 cells are added. Adherent cells are quantified using a Nikon TE300 fluorescent microscope. In some wells, 1000U DNAse1 is added 10 min before counting to degrade NETs

Introduction

Neutrophil extracellular traps (NETs) are newly discovered neutrophil-derived elements composed of strands of extracellular DNA decorated by hundreds of proteins and histones. They are secreted by neutrophils in the context of infection and inflammation following specific stimuli and they were initially recognized to be part of the neutrophil’s defense mechanisms against infections. They were first shown to promote trapping of various microbial invaders including bacteria, viruses and fungi ^1-3. Trapping of the microbe would then lead to its destruction both directly by NET-associated proteins ^3,4 and indirectly through local recruitment of....

Protocol

NOTE: All experiments were conducted in accordance with local institutional ethical guidelines.

1. Blood Drawing

1. Through antecubital venipucture, collect 14 tubes of blood from a healthy volunteer into green top (heparinized) tubes and invert each tube before settling on ice. Collect blood within 10 - 15 min of experiment to ensure optimal neutrophil yield.

2. Neutrophil Isolation from Whole Blood

1. Wash each green top tube of blood with 5 ml of PBS without Ca and Mg to dilute the blood. In each of 4 x 50 ml conical tubes, add 15 ml of Lymphocyte Separation Media (LSM) at RT. Usi....

Results

Successful achievement of a static adhesion assay with NETs requires adequate coating of the wells with a monolayer of NETs before the addition of cancer cells (Figure 1). All subsequent steps must be done with care not to disrupt that layer. When an adequate layer of NETs is formed, it is expected to see significant cancer cells adhesion to NETs as seen in Figure 2A and 2C. This effect is abrogated by the addition of DNAse1, which degrades NETs as seen in Figure.......

Discussion

The Neutrophil Extracellular Trap isolation protocol demonstrated in this video combines different techniques used in the literature for neutrophil isolation and NET formation. It simplifies a fairly complex and variable process and offers a very reliable and replicable way to isolate purified cell-free NETs with fewer steps than other protocols. Furthermore, readily available reagents are employed adding to the protocols simplicity and significantly reducing its cost. The application of NETs towards a static adhesion as.......

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
Lymphocyte Separation Medium (LSM)	Wisent	305-010-CL
Dextran hydrochloride (powder)	Spectrum	DE130	6% Dextran solution is made by supplementing PBS with Ca and Mg with 6% Dextran powder
RPMI-1640 Medium with L-glutamine	Wisent	350-000-CL	3% RPMI solution is made by supplementing RPMI with 3% Fetal Bovine serum
BD Pharm Lyse Lysing buffer	BD Biosciences	555899
Phorbol 12-myristate 13-acetate (PMA)	Sigma-Alrdrich	P8139
DNAse1	Roche	11284932001
F12 DMEM	Wisent	319-075-CL
Fetal Bovine Serum (FBS)	Wisent	080-150
Penicillin/Streptomycin	Gibco	15140-122
CFSE	Invitrogen	C1157
0.25% Trypsin-EDTA	Gibco	25200-056
Integrid tissue culture dish with 20 mm grid (150 x 25 mm)	Falcon	353025