High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro

Summary

High throughput assays are presented that in combination provide excellent tools to quantitate NET release from human neutrophils.

Abstract

Neutrophil granulocytes are the most abundant leukocytes in the human blood. Neutrophils are the first to arrive at the site of infection. Neutrophils developed several antimicrobial mechanisms including phagocytosis, degranulation and formation of neutrophil extracellular traps (NETs). NETs consist of a DNA scaffold decorated with histones and several granule markers including myeloperoxidase (MPO) and human neutrophil elastase (HNE). NET release is an active process involving characteristic morphological changes of neutrophils leading to expulsion of their DNA into the extracellular space. NETs are essential to fight microbes, but uncontrolled release of NETs has been associated with several disorders. To learn more about the clinical relevance and the mechanism of NET formation, there is a need to have reliable tools capable of NET quantitation.

Here three methods are presented that can assess NET release from human neutrophils in vitro. The first one is a high throughput assay to measure extracellular DNA release from human neutrophils using a membrane impermeable DNA-binding dye. In addition, two other methods are described capable of quantitating NET formation by measuring levels of NET-specific MPO-DNA and HNE-DNA complexes. These microplate-based methods in combination provide great tools to efficiently study the mechanism and regulation of NET formation of human neutrophils.

Introduction

NET formation is a novel mechanism by which neutrophils fight pathogens.¹ The core of NETs is nuclear DNA.¹ This DNA network is associated with neutrophil granule proteins and histones.¹ The main form of NET formation requires the death of neutrophils characterized by chromatin decondensation, disappearance of granular and nuclear membranes, translocation of neutrophils elastase to the nucleus, citrullination of histones and finally the spill of DNA-based NETs.² NETs entrap and kill a wide variety of microbes and are an essential part of the innate immune weapon repertoire. Uncontrolled NET formation has, however, been linke....

Protocol

The Institutional Review Board of the University of Georgia approved the human subject study to collect peripheral blood from healthy volunteers (UGA# 2012-10769-06).^5,7,8 Volunteers signed the required informed consent form before blood draw. The research performed in this article is in compliance with the ethical guidelines for medical research involving human subjects of the Declaration of Helsinki.

1. Isolation of Neutrophils from Peripheral Human Blood (Figure 1)

Note: There are several ways to isolate neutrophils from peripheral blood. The following protocol provides one possibility. This protocol yields large numbers of non-activated huma....

Results

The figures in this manuscript describe the method of neutrophil isolation, experimental procedures, and present representative results with explanation of data analysis. Figure 1 shows the sequential steps of human neutrophil preparation. This protocol represents only one possible way of neutrophil isolation. It yields large amounts of resting neutrophils capable of releasing NETs upon stimulation. Figure 2 shows how the fluorescence-based DNA release as.......

Discussion

NETs represent a fascinating novel mechanism by which neutrophils kill pathogens.¹ Although the literature of NETs has been continuously expanding over the last ten years since their discovery, several important questions related to their role in biology, mechanism and regulation remain unclear. Appropriate methodology has to be developed to measure NETs, this very unique antimicrobial mechanism. This article describes methods that can be used to quantitate NETs in a high throughput manner. The first assay fol.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Anti-Human Neutrophil Elastase  Rabbit Ab	Calbiochem	481001	1:2,000x coated
Anti-Myeloperoxidase Ab (Rabbit)	Millipore	07-496	1:2,000x coated
DNase-1	Roche	10-104-159-001	1 µg/ml used for digestion
20 mM EGTA/ PBS	Sigma-Aldrich	E3889-25G
2.5 mM EGTA/PBS	Sigma-Aldrich	E3889-25G
Cell death detection ELISA Anti-DNA POD	Roche	11544675001	1:500x
Eon Microplate Spectrophotometer	Biotek
Gen5 All-in-One microplate software	Biotek		analytical tool (ELISA)
Sytox orange	Life Technology	S11368	0.2% final concentration/volume
1 M Hepes	Cellgro	25-060-Cl	Use 10 mM final concentration.
1 M glucose	Sigma		Use 5 mM final concentration.
HBSS	Corning	21-023-CM
Varioskan Flash Ver.2.4.3	Thermoscientific
PMA	Sigma	P 8139	100 nM final used
ELISA Plate	Greiner bio-one	655061
Conical tubes 15 ml	Thermoscientific	339650
Conical tubes 50 ml	Thermoscientific	339652
Percoll (pH 8.5-9.5)	Sigma	P 1644	Sodium Chloride, Sigma, S7653-250G
Dextran	Spectrum	D1004
RPMI 1640 media	Corning Cellgro	17-105-CV
96 well assay plate black plate clear bottom	Costar	3603