Name: high throughput measurement of extracellular dna release and quantitative net formation in human neutrophils in vitro
Uploaded: 2016-06-18T13:00:00.000+00:00
Duration: 11 min 3 s
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.

조지아 대학의 임상 시험 심사위원회는 건강한 지원자 (UGA 번호 2012-10769-06)에서 말초 혈액을 수집하는 인간 대상 연구를 승인했다. 5,7,8 자원 봉사자 혈액 그리기 전에 필요한 정보 동의서에 서명했다. 이 문서에서 수행 된 연구는 헬싱키 선언의 인체를 이용한 의학 연구에 대한 윤리 지침을 준수합니다. 주변 인간의 혈액에서 호중구의 1. 분리 (그림 1) 주 : 말초 혈액 호중구를 분리하는 방법에는 여러 가지가있다. 다음 프로토콜은 하나의 가능성을 제공합니다. 이 프로토콜은 외부 자극에 그물을 방출 할 수있는 비 - 활성화 인간 호중구 다수 산출한다. 40 ml의 정맥 천자에 의한 건강한 지원자에서 얻은 전체 혈액을 사용합니다. 7,9 <ol><li> 두 50 ML 원뿔 튜브로 나누어지는 20 ml의 혈액입니다. 10 ml의 6 % 덱스 트란을 추가합니다. 부드럽게 섞는다. </li><li> 20 분 이전 백혈구 후에 RIC깨끗한 50 ML 원뿔 튜브에 어떤 펠렛 적혈구를 복용하고, 멸균 PBS로를 작성하지 않고 시간 상위 단계입니다. </li><li> 400 XG에 10 분 원심 분리기. 4 용액에 재현 탁 펠렛은 PBS를 멸균. </li><li> 85 %의 5 단계 퍼콜 구배를 준비 -80 % -75 % -70 % -65 %이 15 ML 원뿔 튜브 및 레이어 2 ㎖의 백혈구 서스펜션의 각 그라데이션의 상단에. </li><li> 브레이크 해제와 함께 30 분 동안 원심 분리기 800 XG. </li><li> 모든 셀 75 % / 80 %에서 레이어 (호중구) 70 % / 75 % 인터페이스를 수집합니다. </li><li> PBS에 두 번 (350 XG, 10 분, RT)을 세포를 씻으과 혈구를 사용하여 세포를 계산합니다. </li></ol> 마이크로 플레이트 형광 계를 사용하여 세포 외 DNA 자료의 속도론 2. 측정 참고 :이 방법은 막 불 투과성 DNA 결합 96 웰 마이크로 포맷에 DNA 자료를 나타내는 염료 (그림 2)의 형광의 변화를 측정 할 수 있습니다. <ol><li> + 분석 매체에서 호중구의 현탁액 (HBSS 준비2 × 106 세포 / ml의 농도에서 1 % (v / v)의자가 혈청 + 5 mM의 포도당). </li><li> 막 투과성 DNA 결합 염료의 5 μmol / L를 추가합니다. 부드럽게 섞는다. </li><li> 나누어지는 인간 호중구 (50 μL / 웰) 마이크로 96 웰 블랙, 투명 바닥 위에. 확인 분석 배지를 웰의 전체 바닥을 포함합니다. 그렇지 않은 경우, 측면에 부드럽게 판을 누릅니다. </li><li> 37 ° C에서 10 분간 플레이트 포함 호중구을 따뜻하게. </li><li> 한편 최종 농도의 두 배에 자극의 솔루션을 준비하는 것은 사용되는 (37 ° C 따뜻한 분석 매체에서). 긍정적 인 NET 컨트롤이 최대 NET 자료를 트리거하기 위해 4 시간 동안 100 나노 포르 - 미리 스테이트 아세테이트 (PMA)와 인간 호중구를 사용할 때 자발적 NET 형성이 낮은 5,8 남아있다. 4 시간 동안 5,8 PMA 자극은 최대 NET 자료를 보장합니다. </li><li> 측정 (4 시간, 37 ° C, 여기에 대한 530 nm의 및 배출을위한 590 ㎚)에 대한 마이크로 형광 계를 준비​​합니다. &lt;/ 리&gt; <li> 50 μL 호중구 현탁액 50 μL 자극 용액을 첨가하여 호중구를 자극한다. 최대 DNA 해제 신호를 측정하기 위해 잘 한 0.5 μg의 / ㎖ 사포닌을 사용합니다. </li><li> 리더 플레이트를 놓고없이 진탕 매 2 분에서 4 시간 동안 형광의 변화를 측정한다. </li><li> 데이터 분석을 위해, 시간에 따른 형광의 증가 정규화를 계산한다. <ol><li> 사포닌을 포함한 모든 우물 용 엔드 포인트 값에서 빼기 기준 형광 (그림 2A, B). 사포닌 형광 상승이 가장 높은 신호이어야한다. 이것은 &quot;DNA 최대 방출&quot;(도 2A)로 지칭된다. </li><li> 최대 DNA 릴리스 미지 시료의 형광의 증가를 분할하여 미지 시료에서 DNA 방출의 비율을 계산합니다. </li><li> 복제의 평균 결과는 &quot;최대 DNA의 출시 %&quot;(그림 2C)로 제시. </li></ol></li></ol> NET 양식 3. 정량MPO-DNA와 HNE-DNA ELISA 분석 실험에 의해 ATION 참고 : 우리의 실험실에서이 분석 수정 (MPO-DNA) 또는 설립 (HNE-DNA)를 MPO-DNA와 HNE-DNA 복합체의 수준을 측정하여 NET 형성을 정량 5. <ol><li> 코트 96 웰 높은 결합 하룻밤 캡처 항체 (50 μL / 웰) 용량 ELISA 플레이트 : 안티 - MPO (1 : 2,000 희석) 또는 안티 HNE (1 : 2,000). </li><li> PBS로 세척 ELISA 플레이트는 세 번 (200 μL / 웰)와 RT에서 2 시간 동안 5 % BSA 및 0.1 % 인간 혈청 알부민을 블록 (200 μL / 웰). PBS로 3 회 세척 할 것. </li><li> 시드 96 웰 마이크로 / 웰로 100 ㎕를 분석 배지 중 100,000 세포의 밀도로 호중구 및 NET 형성을 자극한다. 세포를 품어 : 37 ° C 4 시간. </li><li> 그들을 잘 혼합, 호중구 상층 액 2 U / ㎖ DNase를 추가하여 제한된 DNase의 소화를 수행합니다. 실온에서 보관하십시오. </li><li> 11 μL 25 밀리미터 EGTA (최종 농도 2.5 mm)를 추가하여 15 분 후 DNase의를 중지합니다. 잘 섞다. 스와 수집청소의 microfuge 튜브 pernatants. 스핀 샘플 RT에서 5 분 동안 300 XG에서 세포 파편을 제거한다. 청소의 microfuge 튜브에 자신의 상층 액을 전송합니다. 준비가 될 때까지 얼음에 보관하십시오. </li><li> 이전에 준비 &quot;NET 표준&quot;의 나누어지는을 사용합니다. 주 : NET-표준은 적어도 5 개의 다른 독립적 인 인간 공여자로부터 수득 PMA 자극 호중구의 DNase 소화 상등액의 혼합이다. <ol><li> 오랫동안 동일한 표준을 사용하여 수집하고 각 공여자로부터 호중구 상청액 대량 나누어지는한다. 예를 들어 PMA 자극 호중구의 DNase의 소화 상층 액을 2 ㎖를 수집하는 것은 200 분량 씩 200 ELISA 플레이트에 충분한 (10 μL 각)가 발생합니다. 인터넷 표준을 특성화 데이터는 그림 5에서 발견된다. </li><li> 4 시간 동안 100 nm의 PMA와 인간 호중구를 자극하고 DNase의이 단계 3.4-3.5있어서 자신의 상층 액을 소화 적용됩니다. </li><li> 수집, 수영장, 나누어지는 (10 μL) 무료ZE (-20 ° C) 호중구 상층 액. </li><li> 적어도 5 개의 개별 기증자로부터 획득 한 나누어지는 / 기증자 해동, &quot;표준 NET의&quot;를 준비하려면를 혼합하고 얼음에 보관하십시오. </li><li> 해동 샘플을 취소하고 다음 실험을 위해 신선한 것을 사용합니다. </li><li> PBS + EGTA에서 NET-표준의 2 용액을 희석 : 1을 준비합니다. </li></ol></li><li> DNase1 소화 시료 (표준 및 알 수없는 상층 액) PBS + EGTA 20 배 희석 캡처 항체로 코팅 된 ELISA 플레이트에 그들을 나누어지는. </li><li> 4 ° C에서 밤새 ELISA 플레이트를 품어 PBS로 세 번 씻는다. </li><li> 검출 항체 용액을 적용 (100 μL / 웰) : 말 래 디쉬 퍼 옥시다아제 접합 항 DNA 항체 (1 : 500, 마우스). 어둠 속에서 1 시간 동안 품어. </li><li> PBS와 네 세척 후 TMB 퍼 옥시 다제 기판을 추가 : 100 μL / 웰, 30 분. 블루 착색은 퍼 옥시 다제 활성 (현재 그물)에 대한 표시입니다. </li><li> 잘 100 μL / 1 M 염산을 첨가하여 반응을 중지합니다. 그만큼블루 솔루션은 노란색 (그림 5A)를 설정합니다. </li><li> 마이크로 광도계를 사용하여 450 nm에서 흡광도를 참조하십시오. </li><li> 데이터 분석을 위해, NET 표준에 비해 MPO-DNA 또는 HNE-DNA 복합체의 양을 계산한다. <ol><li> 모든 샘플에서 분석 배지의 배경 광 밀도 값을 뺀다. </li><li> 상대 NET 콘텐츠 (Y 축) (도 5a)에 대한 희석하여 표준 시료의 흡광도 값 (X 축)을 그린다. </li><li> 이 표준 곡선의 nonsaturated 범위를 사용하면 (그림 5A) (사용되는 소프트웨어에서 최상의 지수 맞게 사용) 추세선을 설정합니다. 이 추세선 방정식 그물의 양을 양적하기 OD 값의 변환을 결정한다. </li><li> 인터넷 표준 (그림 5A)의 비율로 미지 시료에 그물의 양을 줄 것이다 3.13.3에서 추세 줄 식으로 미지의 측정 된 OD 값을 삽입합니다. </li><li> 계산하다복제 (삼중)의 평균 (그림 5C) &quot;MPO-DNA 또는 HNE-DNA 복합체 (표준의 %)의 양&quot;으로 최종 데이터를 제시한다. </li></ol></li></ol>

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The authors have nothing to disclose.

그물 그물의 문학은 지속적으로, 생물학,기구 및 규제에서의 역할에 관한 몇 가지 중요한 질문이 불분명 남아 자신의 발견 이후 지난 10 년에 걸쳐 확대되고 있지만 병원균을 죽인다. 1 호중구하는 매혹적인 새로운 메커니즘을 나타냅니다. 적절한 방법은 그물이 매우 독특한 항균 메카니즘을 측정하기 위해 개발되어야한다. 이 문서는 높은 처리량 방식으로 그물을 정량하는 데 사용할 수?...

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><tbody><tr><td>Anti-Human Neutrophil Elastase&amp;nbsp; Rabbit Ab&amp;nbsp;</td><td>Calbiochem</td><td>481001</td><td>1:2,000x coated</td></tr><tr><td>Anti-Myeloperoxidase Ab (Rabbit)</td><td>Millipore</td><td>07-496</td><td>1:2,000x coated</td></tr><tr><td>DNase-1</td><td>Roche</td><td>10-104-159-001</td><td>1 &amp;#181;g/ml used for digestion</td></tr><tr><td>20 mM 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&amp;nbsp;</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>100 nM final used</td></tr><tr><td>ELISA Plate</td><td>Greiner bio-one</td><td>655061</td><td></td></tr><tr><td>Conical tubes 15 ml</td><td>Thermoscientific</td><td>339650</td><td></td></tr><tr><td>Conical tubes 50 ml</td><td>Thermoscientific</td><td>339652</td><td></td></tr><tr><td>Percoll (pH 8.5-9.5)&amp;nbsp;</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.

이 원고의 수치. 호중구 격리, 실험 절차 및 데이터 분석의 설명과 함께 현재 대표적인 결과의 방법을 서술 한 인간 호중구 준비의 순차적 인 단계를 보여줍니다. 이 프로토콜은 호중구 격리의 하나의 가능한 방법을 나타냅니다. 그것은 자극에 그물을 방출 할 수있는 호중구 휴식의 많은 양을 산출한다. 형광 기반의 DNA 자료 분석의 작동 방식 <stron...

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.

세포 외 DNA 릴리스의 높은 처리량 측정 및 인간 호중구의 양적 NET 형성<em&gt; 체외</em

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

high-throughput-measurement-extracellular-dna-release-quantitative

Research

JoVE Journal

Immunology and Infection

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

13.0K 조회수.  University of Georgia. High throughput assays are presented that in combination provide excellent tools to quantitate NET release from human neutrophils.

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

Direct Observation of Phagocytosis and NET-formation by Neutrophils in Infected Lungs using 2-photon Microscopy

After the gastrointestinal tract, the lung is the second largest surface for interaction between the vertebrate body and the 
environment. Here, an effective gas exchange must be maintained, while at the same time avoiding infection by the multiple pathogens that are inhaled 
during normal breathing. To achieve this, a superb set of defense strategies combining humoral and cellular immune mechanisms exists. One of the most 
effective measures for acute defense of the lung is the recruitment of neutrophils, which either phagocytose the inhaled pathogens or kill them by 
releasing cytotoxic chemicals. A recent addition to the arsenal of neutrophils is their explosive release of extracellular DNA-NETs by which bacteria 
or fungi can be caught or inactivated even after the NET releasing cells have died. We present here a method that allows one to directly observe neutrophils, 
migrating within a recently infected lung, phagocytosing fungal pathogens as well as visualize the extensive NETs that they have produced throughout the 
infected tissue. The method describes the preparation of thick viable lung slices 7 hours after intratracheal infection of mice with conidia of the mold 
Aspergillus fumigatus and their examination by multicolor time-lapse 2-photon microscopy. This approach allows one to directly investigate antifungal 
defense in native lung tissue and thus opens a new avenue for the detailed investigation of pulmonary immunity.

We show, how to use 2-photon microscopy for the observation of the dynamics of neutrophil granulocytes in infected lungs while they phagocytose pathogens or produce neutrophil extracellular traps (NETs).

2 광자 현미경을 사용하여 감염된 폐의 Neutrophils에 의해 Phagocytosis와 NET - 형성의 직접 관측

After the gastrointestinal tract, the lung is the second largest surface for interaction between the vertebrate body and the 
environment. Here, an ...

연구

면역학 및 감염병학

Simplified Human Neutrophil Extracellular Traps (NETs) Isolation and Handling

Neutrophil Extracellular Traps (NETs) have been recently identified as part of the neutrophil&#8217;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

Simplified Human Neutrophil Extracellular Traps NETs Isolation and Handling

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.

단순화 된 인간 호중구 세포 외 트랩 (그물)의 분리 및 처리

Neutrophil Extracellular Traps (NETs) have been recently identified as part of the neutrophil&#8217;s antimicrobial armamentarium. Apart from their role ...

A Simple Fluorescence Assay for Quantification of Canine Neutrophil Extracellular Trap Release

Neutrophil extracellular traps are networks of DNA, histones and neutrophil proteins released in response to infectious and inflammatory stimuli. Although a component of the innate immune response, NETs are implicated in a range of disease processes including autoimmunity and thrombosis. This protocol describes a simple method for canine neutrophil isolation and quantification of NETs using a microplate fluorescence assay. Blood is collected using conventional venipuncture techniques. Neutrophils are isolated using dextran sedimentation and a density gradient using conditions optimized for dog blood. After allowing time for attachment to the wells of a 96 well plate, neutrophils are treated with NET-inducing agonists such as phorbol-12-myristate-13-acetate or platelet activating factor. DNA release is measured by the fluorescence of a cell-impermeable nucleic acid dye. This assay is a simple, inexpensive method for quantifying NET release, but NET formation rather than other causes of cell death must be confirmed with alternative methods.

Neutrophil extracellular traps (NETs) are networks of DNA, histones and neutrophil proteins. Although a component of the innate immune response, NETs are implicated in autoimmunity and thrombosis. This protocol describes a simple method for canine neutrophil isolation and quantification of NETs using a microplate fluorescence assay.

개 호중구 세포 외 트랩 릴리스의 정량화를위한 ​​간단한 형광 분석

Neutrophil extracellular traps are networks of DNA, histones and neutrophil proteins released in response to infectious and inflammatory stimuli. Although ...

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

In response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with histones and antimicrobial proteins. Excessive NET formation (NETosis) and citH3 release during sepsis is associated with multiple organ dysfunction and mortality in mice and humans but its implications in dogs are unknown. Herein, we describe a technique to isolate canine neutrophils from whole blood for observation and quantification of NETosis. Leukocyte-rich plasma, generated by dextran sedimentation, is separated by commercially available density gradient separation media and granulocytes collected for cell count and viability testing. To observe real-time NETosis in live neutrophils, cell permeant and cell impermeant fluorescent nucleic acid stains are added to neutrophils activated either by lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA). Changes in nuclear morphology and NET formation are observed over time by fluorescence microscopy. In vitro NETosis is further characterized by co-colocalization of cell-free DNA (cfDNA), myeloperoxidase (MPO) and citrullinated histone H3 (citH3) using a modified double-immunolabelling protocol. To objectively quantify NET formation and citH3 expression using fluorescence microscopy, NETs and citH3-positive cells are quantified in a blinded manner using available software. This technique is a specific assay to evaluate the in vitro capacity of canine neutrophils to undergo NETosis.

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

We describe methods to isolate canine neutrophils from whole blood and visualize NET formation in live neutrophils using fluorescence microscopy. Also described are protocols to quantify NET formation and citrullinated histone H3 (citH3) expression using immunofluorescence microscopy.

생체 외에서 형광 현미경 검사 법에 의해 개 호 중구 Extracellular 함정 형성: 역동적이 고 양적 분석

In response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with ...

Automated Image-Based Quantification of Neutrophil Extracellular Traps Using NETQUANT

Neutrophil extracellular traps (NETs) are web-like antimicrobial structures consisting of DNA and granule derived antimicrobial proteins. Immunofluorescence microscopy and image-based quantification methods remain important tools to quantitate neutrophil extracellular trap formation. However, there are key limitations to the immunofluorescence-based methods that are currently available for quantifying NETs. Manual methods of image-based NET quantification are often subjective, prone to error and tedious for users, especially non-experienced users. Also, presently available software options for quantification are either semi-automatic or require training prior to operation. Here, we demonstrate the implementation of an automated immunofluorescence-based image quantification method to evaluate NET formation called NETQUANT. The software is easy to use and has a user-friendly graphical user interface (GUI). It considers biologically relevant parameters such as an increase in the surface area and DNA:NET marker protein ratio, and nuclear deformation to define NET formation. Furthermore, this tool is built as a freely available app, and allows for single-cell resolution quantification and analysis.

Here, we present a protocol for generating neutrophil extracellular traps (NETs) and operating NETQUANT, a fully automatic software option for quantification of NETs in immunofluorescence images.

NETQUANT를 사용한 호중구 세포외 트랩의 자동 이미지 기반 정량화

Neutrophil extracellular traps (NETs) are web-like antimicrobial structures consisting of DNA and granule derived antimicrobial proteins. ...

A High-throughput Assay to Assess and Quantify Neutrophil Extracellular Trap Formation

Neutrophil extracellular traps (NETs) are immunogenic extracellular DNA structures that can be released by neutrophils upon a wide variety of triggers. NETs have been demonstrated to serve as an important host defense mechanism that traps and kills microorganisms. On the other hand, they have been implicated in diverse systemic autoimmune diseases. NETs are immunogenic and toxic structures that contain a pool of relevant autoantigens including anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) and systemic lupus erythematosus (SLE). Different forms of NETs can be induced depending on the stimulus. The amount of NETs can be quantified using different techniques including measuring DNA release in supernatants, measuring DNA-complexed with NET-molecules like myeloperoxidase (MPO) or neutrophil elastase (NE), measuring the presence of citrullinated histones by fluorescence microscopy, or flow cytometric detection of NET-components which all have different features regarding their specificity, sensitivity, objectivity, and quantity. Here is a protocol to quantify ex vivo NET formation in a highly-sensitive, high-throughput manner by using three-dimensional immunofluorescence confocal microscopy. This protocol can be applied to address various research questions about NET formation and degradation in health and disease.

This protocol describes a highly sensitive and high throughput neutrophil extracellular trap (NET) assay for the semi-automated quantification of ex vivo NET formation by immunofluorescence three-dimensional confocal microscopy. This protocol can be used to evaluate NET formation and degradation after different stimuli and can be used to study potential NET-targeted therapies.

평가 하 고 호 중구 Extracellular 함정 대형 계량 높은 처리량 분석 결과

Neutrophil extracellular traps (NETs) are immunogenic extracellular DNA structures that can be released by neutrophils upon a wide variety of triggers. ...

Quantifying Myeloperoxidase-DNA and Neutrophil Elastase-DNA Complexes from Neutrophil Extracellular Traps by Using a Modified Sandwich ELISA

Certain stimuli, such as microorganisms, cause neutrophils to release neutrophil extracellular traps (NETs), which are basically web-like structures composed of DNA with granule proteins, such as myeloperoxidase (MPO) and neutrophil elastase (NE), and cytoplasmic and cytoskeletal proteins. Although interest in NETs has increased recently, no sensitive, reliable assay method is available for measuring NETs in clinical settings. This article describes a modified sandwich enzyme-linked immunosorbent assay to quantitatively measure two components of circulating NETs, MPO-DNA and NE-DNA complexes, which are specific components of NETs and are released into the extracellular space as breakdown products of NETs. The assay uses specific monoclonal antibodies for MPO or NE as the capture antibodies and a DNA-specific detection antibody. MPO or NE binds to one site of the capture antibody during the initial incubation of samples containing MPO-DNA or NE-DNA complexes. This assay shows good linearity and high inter-assay and intra-assay precision. We used it in 16 patients with COVID-19 with accompanying acute respiratory distress syndrome and found that the plasma concentrations of MPO-DNA and NE-DNA were significantly higher than in the plasma obtained from healthy controls. This detection assay is a reliable, highly sensitive, and useful method for investigating the characteristics of NETs in human plasma and culture supernatants.

We present a protocol for a modified sandwich enzyme-linked immunosorbent assay technique to quantitatively measure two components of neutrophil extracellular trap remnants, myeloperoxidase conjugated-DNA and neutrophil elastase conjugated-DNA complexes,derived from activated neutrophils.

변형된 샌드위치 ELISA를 사용하여 호중구 세포외 트랩에서 Myeloperoxidase-DNA 및 Neutrophil Elastase-DNA 복합체 정량화

Certain stimuli, such as microorganisms, cause neutrophils to release neutrophil extracellular traps (NETs), which are basically web-like structures ...

Dual-Dye Live Cell Analysis를 사용한 NET 형성 정량화

Real-Time High Throughput Technique to Quantify Neutrophil Extracellular Traps Formation in Human Neutrophils

Neutrophils are myeloid-lineage cells that form a crucial part of the innate immune system. The past decade has revealed additional key roles that neutrophils play in the pathogenesis of cancer, autoimmune diseases, and various acute and chronic inflammatory conditions by contributing to the initiation and perpetuation of immune dysregulation through multiple mechanisms, including the formation of neutrophil extracellular traps (NETs), which are structures crucial in antimicrobial defense. Limitations in techniques to quantify NET formation in an unbiased, reproducible, and efficient way have restricted our ability to further understand the role of neutrophils in health and diseases. We describe an automated, real-time, high-throughput method to quantify neutrophils undergoing NET formation using a live cell imaging platform coupled with a membrane permeability-dependent dual-dye approach using two different DNA dyes to image intracellular and extracellular DNA. This methodology is able to help assess neutrophil physiology and test molecules that can target NET formation.

저자 스포트라이트: Quantifying Neutrophil Extracellular Traps in Disease and Drug Screening Using Dual-Color Live-Cell Imaging(이중 색상 생세포 이미징을 사용한 질병 및 약물 스크리닝에서 호중구 세포외 트랩 정량화)

We present an automated high-throughput method to quantify neutrophil extracellular traps (NETs) utilizing the live cell analysis system, coupled with a membrane permeability-dependent dual-dye approach.

세포 외 DNA 릴리스의 높은 처리량 측정 및 인간 호중구의 양적 NET 형성

요약

더 많은 비디오 탐색

이 비디오의 챕터

2 광자 현미경을 사용하여 감염된 폐의 Neutrophils에 의해 Phagocytosis와 NET - 형성의 직접 관측

단순화 된 인간 호중구 세포 외 트랩 (그물)의 분리 및 처리

개 호중구 세포 외 트랩 릴리스의 정량화를위한 간단한 형광 분석

생체 외에서 형광 현미경 검사 법에 의해 개 호 중구 Extracellular 함정 형성: 역동적이 고 양적 분석

NETQUANT를 사용한 호중구 세포외 트랩의 자동 이미지 기반 정량화

평가 하 고 호 중구 Extracellular 함정 대형 계량 높은 처리량 분석 결과

변형된 샌드위치 ELISA를 사용하여 호중구 세포외 트랩에서 Myeloperoxidase-DNA 및 Neutrophil Elastase-DNA 복합체 정량화

인간 호중구에서 호중구 세포외 트랩 형성을 정량화하기 위한 실시간 고처리량 기법

인간 호중구에서 호중구 세포외 트랩 형성을 정량화하기 위한 실시간 고처리량 기법

인간 호중구에서 호중구 세포외 트랩 형성을 정량화하기 위한 실시간 고처리량 기법

세포 외 DNA 릴리스의 높은 처리량 측정 및 인간 호중구의 양적 NET 형성

요약

더 많은 비디오 탐색

이 비디오의 챕터

2 광자 현미경을 사용하여 감염된 폐의 Neutrophils에 의해 Phagocytosis와 NET - 형성의 직접 관측

단순화 된 인간 호중구 세포 외 트랩 (그물)의 분리 및 처리

개 호중구 세포 외 트랩 릴리스의 정량화를위한 ​​간단한 형광 분석

생체 외에서 형광 현미경 검사 법에 의해 개 호 중구 Extracellular 함정 형성: 역동적이 고 양적 분석

NETQUANT를 사용한 호중구 세포외 트랩의 자동 이미지 기반 정량화

평가 하 고 호 중구 Extracellular 함정 대형 계량 높은 처리량 분석 결과

변형된 샌드위치 ELISA를 사용하여 호중구 세포외 트랩에서 Myeloperoxidase-DNA 및 Neutrophil Elastase-DNA 복합체 정량화

인간 호중구에서 호중구 세포외 트랩 형성을 정량화하기 위한 실시간 고처리량 기법

인간 호중구에서 호중구 세포외 트랩 형성을 정량화하기 위한 실시간 고처리량 기법

인간 호중구에서 호중구 세포외 트랩 형성을 정량화하기 위한 실시간 고처리량 기법

개 호중구 세포 외 트랩 릴리스의 정량화를위한 간단한 형광 분석