Name: high throughput measurement of extracellular dna release and quantitative net formation in human neutrophils in vitro
Uploaded: 2016-06-18T13:00:00
Description: Watch this Scientific Journal Video about High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro at JoVE.com

Special thanks to the personnel of the University of Georgia Health Center laboratory for their continuous support of our work on isolating human neutrophils. This work was supported by the start-up fund of Dr. Rada provided by UGA Office of Vice President for Research.

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 t...

<ol>
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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...

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 linked to numerous autoinflammatory diseases.3,4 Despite their increasingly established relevance, little is known about the mechanism and regulation of NET release. 
Neutrophils dying by releasing NETs are different from apoptotic or necrotic neutrophils.3,5 NET-releasing neutrophils show several features that are characteristic for NET formation. Granule components are associated with DNA in NETs.1 Myeloperoxidase (MPO) and human neutrophil elastase (HNE) are both found in primary granules in resting cells but are translocated to the nucleus to bind to DNA in NETs.1 MPO-DNA and HNE-DNA complexes are specific for NETs, do not occur in apoptotic or necrotic neutrophils.1,3,5 Chromatin decondensation is another feature typical for NETosis.2 NET release also requires citrullination of histones by peptidyl aminidase 4 (PAD4).6 Citrullinated histones are hallmarks of neutrophils that underwent NET release.6
Here three methods are introduced that in combination provide excellent tools to quantitate NETs on a high-throughput scale. The first assay has been used on the field with different changes and quantitates extracellular DNA release in a microplate format. The second and third assays provide confirmation of NETs by measuring NET-specific MPO-DNA and HNE-DNA complexes.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Anti-Human Neutrophil Elastase&#160; Rabbit Ab&#160;</td>
			<td>Calbiochem</td>
			<td>481001</td>
			<td>1:2000X coated</td>
		</tr>
		<tr>
			<td>Anti-Myeloperoxidase Ab (Rabbit)</td>
			<td>Millipore</td>
			<td>07-496</td>
			<td>1:2000X coated</td>
		</tr>
		<tr>
			<td>DNase-1</td>
			<td>Roche</td>
			<td>10-104-159-001</td>
			<td>1ug/ml used for digestion</td>
		</tr>
		<tr>
			<td>20mM EGTA/ PBS</td>
			<td>Sigma-Aldrich</td>
			<td>E3889-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>2.5 mM EGTA/PBS</td>
			<td>Sigma-Aldrich</td>
			<td>E3889-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell death detection ELISA Anti-DNA POD</td>
			<td>Roche</td>
			<td>11544675001</td>
			<td>1:500X&#160;</td>
		</tr>
		<tr>
			<td>Eon Microplate Spectrophotometer</td>
			<td>Biotek</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gen5 All-in-One microplate software</td>
			<td>Biotek</td>
			<td></td>
			<td>analytical tool (ELISA)</td>
		</tr>
		<tr>
			<td>Sytox orange</td>
			<td>Life Technology</td>
			<td>S11368</td>
			<td>0.2% final concentration/volume</td>
		</tr>
		<tr>
			<td>1 M Hepes</td>
			<td>Cellgro</td>
			<td>25-060-Cl</td>
			<td>Use 10 mM final concentration.</td>
		</tr>
		<tr>
			<td>1 M glucose</td>
			<td>Sigma</td>
			<td></td>
			<td>Use 5 mM final concentration.</td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Corning</td>
			<td>21-023-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>Varioskan Flash Ver.2.4.3</td>
			<td>Thermoscientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PMA</td>
			<td>Sigma</td>
			<td>P 8139</td>
			<td>100nM final used</td>
		</tr>
		<tr>
			<td>ELISA Plate</td>
			<td>Greiner bio-one</td>
			<td>655061</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical tubes 15ml</td>
			<td>Thermoscientific</td>
			<td>339650</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical tubes 50ml</td>
			<td>Thermoscientific</td>
			<td>339652</td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll (pH 8.5-9.5)&#160;</td>
			<td>Sigma</td>
			<td>P 1644</td>
			<td>Sodium Chloride, Sigma, S7653-250G</td>
		</tr>
		<tr>
			<td>Dextran</td>
			<td>Spectrum</td>
			<td>D1004</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 media</td>
			<td>Corning Cellgro</td>
			<td>17-105-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well assay plate black plate clear bottom</td>
			<td>Costar</td>
			<td>3603</td>
			<td></td>
		</tr>
	</tbody>
</table>

high throughput measurement of extracellular dna release and quantitative net formation in human neutrophils in vitro

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.

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...

Watch this Scientific Journal Video about High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro at JoVE.com

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

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

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

Neutrophil granulocytes are the most abundant leukocytes in the human blood. Neutrophils are the first to arrive at the site of infection. Neutrophils ...

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