1. 인간의 혈액에서 PMN 격리 항응고제로 EDTA (에틸렌 다이아 민 테트라 초산) 또는 헤파린 (10 U / ML) 24 ML 인간의 혈액에 대해 사용합니다. <ol><li> 15 ML 팔콘 튜브 6 ML Histopaque 1119를 추가하고 위에 조심스럽게 레이어 5-7 ML 전체 혈액. </li><li> 제동없이 800 XG에서 20 분 동안 원심 분리기. </li><li> 기음과 폐기 노란 맑은 상위 레이어를 신선한 팰컨 튜브에 granulocytes를 포함하는 낮은 붉은 단계를 전송합니다. </li><li> 300 x G. 10 분 PBS 및 원심 분리기와 팔콘 튜브를 작성하여 세포를 씻어 </li><li> 한편 2 ML 10X PBS 18 ML Percoll를 혼합하여 100 % Percoll 솔루션을 준비합니다. 1X PBS 85 %, 80 %, 75 %, 70 % 및 65 % Percoll 4 ML 솔루션을 준비와 함께. </li><li> 주문을 감소 서로의 위에 모든 비율이 겹겹이 ML하여 Percoll 기울기 2 팰컨 튜브를 준비합니다. </li><li> 원심 분리가 뜨는을 제거한 후, PBS 4 ML에 쥐똥과 resuspend sedimented 세포를 결합. </li><li> 그라디언트의 각 방향 resuspension의 신중 레이어 2 ML. </li><li> 제동없이 800 XG에서 20 분 동안 원심 분리기. </li><li> 원심 분리 최고 레이어를 제거하고 65% 계층의 대부분 PBMCs와 85 % 계층까지 새로운 팔콘 튜브에 흰색 나머지 타령을 수집 후. </li><li> 300 x G. 10 분 PBS와 원심 분리기와 팔콘 튜브를 작성하여 세포를 씻어 </li><li> PBS의 2ml에 뜨는 및 resuspend sedimented 세포를 (보통&gt; 95 % PMN 아르)를 제거합니다. </li><li> hemocytometer를 사용하여 셀을 계산합니다. </li></ol> 2. 활성화 PMNs <ol><li> 각 우물에 살균 13mm 원형 유리 커버 슬립 (# 1.5)를 넣어 24 웰 세포 배양 판을 준비 </li><li> 씨앗이 500μl RPMI에서 X 10 5 세포를 잘 당 (2 % 인간 혈청 알부민을 포함)과 37 CO 2 배양기에서 1 시간을위한 품어 ° C. </li><li> 한편 RPMI에서 600 NM PMA 솔루션을 준비하고 100μl 잘 따라 세포에 추가할 수 있습니다. 37까지 CO 2 배양기에서 4 H 15 분부터 품어 ° C. </li><li> 4퍼센트 (최종 농도) PBS에 용해 paraformaldehyde에서 세포를 수정. </li></ol> PMA는 긍정적인 제어 역할을하고 지금까지 NET 형성을 유도하는 가장 강력한 대리인이다. 또한, 병원균과 다른 자극 또는 공동 재배는 NET 유도 사용할 수 있습니다. 시간 코스 진행되는 동안 추가 병렬 샘플 준비하는 경우 NET 형성의 각 상태는 확인하실 수 있습니다. Sytox 그린 (Invitrogen)과 같은 비 permeant DNA의 염료가 아닌 고정 셀을 추가하는 경우에만 세포 DNA가 감지됩니다. 새로운 그물 형성이 어떻게든 때마다 포인트, Sytox의 면전에서 장애인이기 때문에 한 병렬 샘플은 사용할 수 있습니다. 3. immunolabeling에 의해 NET 검출 그물도 고정 후 매우 연약하고, 아주 조심스럽게 조작으로 달리 대부분이 준비 중 길을 잃기 쉬우니있다. <ol><li> 신중하게 굽은 바늘로 가장자리에 그것을 리프팅하여 24 잘 문화 플레이트의 부착 세포와 유리 커버 전표를 제거하고 훌륭한 포셉로 점유. PBS의 드롭에 거꾸로 커버 슬립을 넣어. 이 테스트 튜브 스탠드를 덮고 Parafilm 시트에서 할 수 있습니다. 5 분이 3 번 같은 씻으십시오. </li><li> 트리톤은 X - 100 RT 1 분 세포를 permeabilize하기 위해 0.5 %의 드롭 같은 방식으로 커버 전표를 품어. 1 분을위한 PBS로 3 번 씻으십시오. </li><li> Parafilm와 젖은 휴지로 습기 챔버를 준비합니다. 덮개가 차단 버퍼 (5 % 당나귀 혈청)의 드롭 거꾸로 미끄러져 누워 37 30 분 품어 ° C. </li><li> 버퍼를 차단의 기본 항체, 예를 들어 MS 안티 히스톤 및 RB 안티 호중구 엘라스타제을 희석. </li><li> 기본 항체의 드롭에 버퍼를 차단에서 직접 습기 챔버에서 전표를 커버 37 1 H에 대한 품어 전송 ° C. </li><li> PBS로 5 분 3 번 씻으십시오. </li><li> 이차 항체, 예를 들어 DK 안티 MS Cy2와 버퍼를 차단에 DK 방지 RB Cy3를 희석. </li><li> 이차 항체의 드롭에 습기 챔버에 전표를 커버 37 1 H에 대한 품어 전송 ° C. </li><li> PBS로 5 분 3 번 씻으십시오. 얼룩 DNA는 UV 여기거나 멀리 붉은 여기에 대한 Draq5로 필요하고 디스트로 두 번 씻어 것입니다 Hoechst 33342 (1 μg / ML)와 중 5 분 들어, 형광 현미경 옵션에 따라 다릅니다. 물. </li><li> 유리 슬라이드에 Mowiol의 20μl 방울을 설정하고 거꾸로 전지 커버 전표를 탑재합니다. 세포는 커버 슬립 및 슬라이드 사이에 있어야합니다. Mowiol의 드롭 커버 슬립이 드롭 위치는 균일한 두께의 얇은 레이어를 형성합니다. 일반적으로 샘플을 눌러 필요가 없습니다. 가 아닌 침지 렌즈를 사용하려는 경우, 표본은 조사를 위해 준비가 된 것입니다.침지 렌즈와 현미경의 분석을 위해, Mowiol이 샘플의 가장자리 경화까지 약 1 시간에 대한 표본은 건조하게. </li></ol> 4. 전자 현미경을 검사에 대비하여 그물 (SEM) <ol><li> 2,5 %의 글루 타 알데히드 24 - 잘 판에있는 유리 커버 전표에서 세포를 수정 게시합니다. </li><li> 24 잘 문화 판의 세포를 포함하는 유리 커버 전표를 제거하고 물 한 방울에 거꾸로 내려놔. 5 분이 3 번 같은 씻으십시오. </li><li> 30 분 0,5 % OsO4과 부화를 포함한 24 웰 세포 배양 플레이트에 다시 커버 전표를 전송합니다. </li><li> 24 잘 문화 판의 세포를 포함하는 유리 커버 전표를 제거하고 물 한 방울에 거꾸로 내려놔. 5 분이 3 번 같은 씻으십시오. </li><li> 30 분 1퍼센트 타닌산과 부화를 포함한 24 웰 세포 배양 플레이트에 다시 커버 전표를 전송합니다. </li><li> 단계를 반복 4.2-4.4 </li><li> 다음과 같은 처방 (5 분 각각)를 통해 탈수 : <ul><li> 30 % 에탄올 </li><li> 50 % 에탄올 </li><li> 70 % 에탄올 </li><li> 80 % 에탄올 </li><li> 90 % 에탄올 </li><li> 100 % 에탄올 </li><li> 100 % 에탄올 </li><li> 100 % 에탄올 </li></ul></li><li> 임계점 건조기 건조 표본으로 전표를 커버 전송합니다. </li><li> 얇은 레이어 증발기를 사용하여 5nm 플라틴 / 탄소 층과 표본의 코트 표면 </li></ol> 5. 대표 결과 : 절연 방식은 일반적으로 95%보다 순도와 unstimulated 가능한 neutrophils를 산출. 자극 후 다른 시간 시점에서 고정되면, immunostaining 프로토콜 NETosis의 평평화 세포, 핵 lobules의 손실, 과립의 손실과 핵 (히스톤 IE)와 세분화된 (즉,의 증가 오버랩로 연결 핵 무결성 중 형태학의 변경 순서를 보여줍니다 호중구 엘라스타제) 염색법. 이 프로토콜은 neutrophils과 병원균의 특정 상호 작용을 분석하는 출발점이 될 수 있습니다. 이 상호 작용은 전자 현미경을 스캐닝을위한 준비 프로토콜을 사용하여보다 자세한 해부를하실 수 있습니다. NET 형광 NET 구성 요소 (청색 = DNA, 빨강 = 히스톤, 녹색 = 호중구 엘라스타제)의 스테인드 자극 neutrophils의 샘플. 이미지 NET 현지화, DNA와 histones의 핵 지방화 및 호중구 엘라스타제에 대한 세분화된 패턴 외에도 보여줍니다. SEM PMA의 자극 비 자극 및 PMA 자극 neutrophils을 보여주는 전자 현미경을 검사합니다. 자극 후, neutrophils 밖으로 평평하게하고 그물을 생산합니다. SEM 그물과 시겔라 그물에 갇힌 시겔라 세균의 높은 해상도의 SEM 이미지.

<ol>
	<li>Brinkmann, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+extracellular+traps+kill+bacteria.">Neutrophil extracellular traps kill bacteria.</a> Science. 303, 1532-1535 .</li><li>Urban, C. F., Reichard, U., Brinkmann, V., Zychlinsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+extracellular+traps+capture+and+kill+Candida+albicans+yeast+and+hyphal+forms.">Neutrophil extracellular traps capture and kill Candida albicans yeast and hyphal forms.</a> Cell Microbiol. 8, 668-676 (2006).</li><li>M, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leishmania+amazonensis+promastigotes+induce+and+are+killed+by+neutrophil+extracellular+traps.">Leishmania amazonensis promastigotes induce and are killed by neutrophil extracellular traps.</a> PNAS. 106, 6748-6753 (2009).</li><li>Buchanan, J. T., Simpson, A. J., Aziz, R. K., Liu, G. Y., Kristian, S. A., Kotb, M., Feramisco, J., Nizet, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNase+expression+allows+the+pathogen+group+A+Streptococcus+to+escape+killing+in+neutrophil+extracellular+traps.">DNase expression allows the pathogen group A Streptococcus to escape killing in neutrophil extracellular traps.</a> Curr Biol. 14, 396-400 (2006).</li><li>Fuchs, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+cell+death+program+leads+to+neutrophil+extracellular+traps.">Novel cell death program leads to neutrophil extracellular traps.</a> J Cell Biol. 176, 231-241 (2007).</li><li>Brinkmann, V., Zychlinsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beneficial+suicide:+why+neutrophils+die+to+make+NETs.">Beneficial suicide: why neutrophils die to make NETs.</a> Nat Rev Microbiol. 5, 577-582 (2007).</li><li>Aga, E., Katschinski, D. M., van Zandbergen, G., Laufs, H., Hansen, B., Muller, K., Solbach, W., Laskay, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+the+spontaneous+apoptosis+of+neutrophil+granulocytes+by+the+intracellular+parasite+Leishmania+major.">Inhibition of the spontaneous apoptosis of neutrophil granulocytes by the intracellular parasite Leishmania major.</a> J Immunol. , 169-898 (2002).</li></ol>

제공된 프로토콜은 상당한 순도에 비 자극 neutrophils의 고립, NET 형성의 유도와 NETosis 중 형태학의 변화의 분석을하실 수 있습니다. 주의 표본을 처리하는 경우, 즉 느슨하게 연결된 그물의 대부분의 손실됩니다 거친 세척 조건을 피하기 위해, 다양한 자극 조건 형성 NET (기간, 자극)의 양은 비교할 수 있습니다. 호중구 / 병원체 상호 작용, 자극의 순서, 다른 면역 세포와 상호 작용 :이 존중에서 제공하는 프로토콜은보다 정교한 시나리오를 분석하는 방법을 확립하기위한 출발점이 될 수 있습니다.

neutrophil extracellular traps: how to generate and visualize them

호중구의 granulocytes는 주변 혈액에 leukocytes의 가장 풍부한 그룹입니다. 전문 phagocytes, 그들은 아마 박테리아와 intracellularly 그들을 죽일 phagosome 그들의 항균 과립 퓨즈. 우리는 neutrophils는 미생물을 죽이는의 추가 방법을 발견 : 활성화시, 그들은 함께 세포 섬유를 형성 과립 단백질과 염색질을 공개 바인딩 병원균 것을. 이 소설의 구조, 또는 호중구 세포 트랩 (그물), 독성 요인을 저하 및 세균 1, 곰팡이 기생충 2 3 죽여. 네츠의 구조적 근간은 DNA이며, 그들은 DNases의 존재에 신속하게 저하됩니다. 따라서, DNases을 표현하는 박테리아는 4 더 악성입니다. TEM, SEM, immunofluorescence 및 라이브 셀 이미징 기술을 결합하여 상호 현미경을 사용하여, 우리는 자극에 따라, neutrophils의 핵은 그 모양을 잃게하고 EU와 heterochromatin이 균질 것을 알 수 있습니다. 나중에 핵 봉투 및 과립 점막은 NET 구성 요소의 혼합을 허용 분해. 마지막으로, 그물은 세포막 나누기로 발표하고 있습니다. 이 세포 죽음 프로그램 (NETosis)은 apoptosis와 괴사와는이며 NADPH 산화 효소 5 반응 산소 종의 생성에 따라 달라집니다. 호중구 세포 트랩은 급성 염증의 사이트에서 풍부한 있습니다. 네츠는 바인드 미생물 즉, 확산을 방지하고, 독성 요인을 저하하고 neutrophils도 자신의 수명이 넘어 자신의 항균 기능을 수행 수 있도록함으로써 병원체를 죽이는 항균 대리인의 높​​은 지방 농도를 보장 타고난 면역 반응의 한 형태로 나타납니다. 증거가 증가하고있다, 그러나, 그 그물은 또한 질병에 관련된 면역 syndromes에서 불임 6 다양합니다. 우리는 주변 사람의 혈액 7 호중구 Granulocytes을 분리하고 그물을 형성하도록 자극하는 방법을 설명합니다. 또한 우리는 빛과 전자 현미경의 그물을 시각화하는 프로토콜이 포함됩니다.

Watch this Scientific Journal Video about Neutrophil Extracellular Traps: How to Generate and Visualize Them at JoVE.com

Neutrophil Extracellular Traps: How to Generate and Visualize Them

호중구 세포 트랩 (그물) 병원성 박테리아, 곰팡이 및 기생충에 대항하는 중요한 타고난 면역 메커니즘입니다. 여기 우리는 인간의 피를에서 호중구 granulocytes을 분리하고 그물을 형성 그들을 활성화하는 방법을 설명합니다. 우리는 빛과 전자 현미경의 그물을 시각화하기 위해 준비 기술을 제시한다.

호중구 세포 트랩 : 그들을 생성 및 시각화하는 방법

Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them ...

neutrophil-extracellular-traps-how-to-generate-and-visualize-them

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Immunology and Infection

86.0K 조회수.  Max Planck Institute for Infection Biology. 호중구 세포 트랩 (그물) 병원성 박테리아, 곰팡이 및 기생충에 대항하는 중요한 타고난 면역 메커니즘입니다. 여기 우리는 인간의 피를에서 호중구 granulocytes을 분리하고 그물을 형성 그들을 활성화하는 방법을 설명합니다. 우리는 빛과 전자 현미경의 그물을 시각화하기 위해 준비 기술을 제시한다.

비디오: Neutrophil Extracellular Traps: How to Generate and Visualize Them

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 ...

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면역학 및 감염병학

Methods to Study Lipid Alterations in Neutrophils and the Subsequent Formation of Neutrophil Extracellular Traps

Lipid analysis performed by high performance thin layer chromatography (HPTLC) is a relatively simple, cost-effective method of analyzing a broad range of lipids. The function of lipids (e.g., in host-pathogen interactions or host entry) has been reported to play a crucial role in cellular processes. Here, we show a method to determine lipid composition, with a focus on the cholesterol level of primary blood-derived neutrophils, by HPTLC in comparison to high performance liquid chromatography (HPLC). The aim was to investigate the role of lipid/cholesterol alterations in the formation of neutrophil extracellular traps (NETs). NET release is known as a host defense mechanism to prevent pathogens from spreading within the host. Therefore, blood-derived human neutrophils were treated with methyl-&#946;-cyclodextrin (M&#946;CD) to induce lipid alterations in the cells. Using HPTLC and HPLC, we have shown that M&#946;CD treatment of the cells leads to lipid alterations associated with a significant reduction in the cholesterol content of the cell. At the same time, M&#946;CD treatment of the neutrophils led to the formation of NETs, as shown by immunofluorescence microscopy. In summary, here we present a detailed method to study lipid alterations in neutrophils and the formation of NETs.

Lipids are known to play an important role in cellular functions. Here, we describe a method to determine the lipid composition of neutrophils, with emphasis on the cholesterol level, by using both HPTLC and HPLC to gain a better understanding of the underlying mechanisms of neutrophil extracellular trap formation.

방법은 지질 호중구의 변경 및 호중구 세포 외 트랩의 후속 형성을 연구하기 위해

Lipid analysis performed by high performance thin layer chromatography (HPTLC) is a relatively simple, cost-effective method of analyzing a broad range of ...

Visualizing Macrophage Extracellular Traps Using Confocal Microscopy

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal microscopy methods to visualize macrophage extracellular traps (METs). These extracellular traps are defined by the presence of extracellular chromatin with co-expression of other components such as granule proteases, citrullinated histones, and peptidyl arginase deiminase (PAD). The expression of METs is generally measured after exposure to a stimulus and compared to un-stimulated samples. Samples are also included for background and isotype control. Cells are analyzed using well-defined image analysis software. Confocal microscopy may be used to define the presence of METs both in vitro and in vivo in lung tissue.

Macrophage extracellular traps are a newly described entity. This article will concentrate on confocal microscopy methods and how they are visualized in vitro and in vivo from lung samples.

Confocal 현미경 검사 법을 사용 하 여 대 식 세포 세포 외 트랩을 시각화

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal ...

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. ...

Identification of Neutrophil Extracellular Traps in Paraffin-Embedded Feline Arterial Thrombi using Immunofluorescence Microscopy

Neutrophil extracellular traps (NETs), composed of cell-free DNA (cfDNA) and proteins like histones and neutrophil elastase (NE), are released by neutrophils in response to systemic inflammation or pathogens. Although NETs have previously been shown to augment clot formation and inhibit fibrinolysis in humans and dogs, the role of NETs in cats with cardiogenic arterial thromboembolism (CATE), a life-threatening complication secondary to hypertrophic cardiomyopathy, is unknown. A standardized method to identify and quantify NETs in cardiogenic arterial thrombi in cats will advance our understanding of their pathological role in CATE. Here, we describe a technique to identify NETs in formaldehyde-fixed and paraffin-embedded thrombi within the aortic bifurcation, extracted during necropsy. Following deparaffinization with xylene, aortic sections underwent indirect heat-induced antigen retrieval. Sections were then blocked, permeabilized, and ex vivo NETs were identified by colocalization of cell-free DNA (cfDNA), citrullinated histone H3 (citH3), and neutrophil elastase (NE) using immunofluorescence microscopy. To optimize the immunodetection of NETs in thrombi, autofluorescence of tissue elements was limited by using an autofluorescence quenching process prior to microscopy. This technique could be a useful tool to study NETs and thrombosis in other species and offers new insights into the pathophysiology of this complex condition.

We describe a method to identify neutrophil extracellular traps (NETs) in formaldehyde-fixed and paraffin-embedded feline cardiogenic arterial thrombi using heat-induced antigen retrieval and a double immunolabeling protocol.

면역형광 현미경을 사용하여 파라핀 내장 펠린 동맥 혈전에서 호중구 세포 외 트랩의 식별

Neutrophil extracellular traps (NETs), composed of cell-free DNA (cfDNA) and proteins like histones and neutrophil elastase (NE), are released by ...

Morphological and Compositional Analysis of Neutrophil Extracellular Traps Induced by Microbial and Chemical Stimuli

Neutrophils function as the first line of cellular defense in an innate immune response by employing diverse mechanisms, such as the formation of neutrophil extracellular traps (NETs). This study analyzes the morphological and compositional changes in NETs induced by microbial and chemical stimuli using standardized in vitro methodologies for NET induction and characterization with human cells. The procedures described here allow the analysis of NET morphology (lytic or non-lytic) and composition (DNA-protein structures and enzymatic activity), and the effect of soluble factors or cellular contact on such characteristics. Additionally, the techniques described here could be modified to evaluate the effect of exogenous soluble factors or cellular contact on NET composition.
The applied techniques include the purification of polymorphonuclear cells from human peripheral blood using a double density gradient (1.079-1.098 g/mL), guaranteeing optimal purity and viability (&#8805; 95%) as demonstrated by Wright's staining, trypan blue exclusion, and flow cytometry, including FSC versus SSC analysis and 7AAD staining. NET formation is induced with microbial (Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans) and chemical (phorbol myristate acetate, HOCl) stimuli, and the NETs are characterized by DNA-DAPI staining, immunostaining for the antimicrobial peptide cathelicidin (LL37), and quantification of enzymatic activity (neutrophil elastase, cathepsin G, and myeloperoxidase). The images are acquired through fluorescence microscopy and analyzed with ImageJ.

Presented here is a protocol for the induction and analysis of in vitro neutrophil extracellular traps (NETs). Quantification of DNA, cathelicidin (LL37), and enzyme activity yielded data that show the variability in the composition and morphology of NETs induced by microbial and chemical stimuli under similar controlled conditions.

미생물 및 화학적 자극에 의해 유도 된 호중구 세포 외 트랩의 형태 학적 및 조성 분석

Neutrophils function as the first line of cellular defense in an innate immune response by employing diverse mechanisms, such as the formation of ...

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 ...

호중구 세포외 트랩을 시각화하기 위한 염색 및 이미징

Immunofluorescence Imaging of Neutrophil Extracellular Traps in Human and Mouse Tissues

Neutrophil extracellular traps (NETs) are released by neutrophils as a response to bacterial infection or traumatic tissue damage but also play a role in autoimmune diseases and sterile inflammation. They are web-like structures composed of double-stranded DNA filaments, histones, and antimicrobial proteins. Once released, NETs can trap and kill extracellular pathogens in blood and tissue. Furthermore, NETs participate in homeostatic regulation by stimulating platelet adhesion and coagulation. However, the dysregulated production of NETs has also been associated with various diseases, including sepsis or autoimmune disorders, which makes them a promising target for therapeutic intervention. Apart from electron microscopy, visualizing NETs using immunofluorescence imaging is currently one of the only known methods to demonstrate NET interactions in tissue. Therefore, various staining methods to visualize NETs have been utilized. In the literature, different staining protocols are described, and we identified four key components showing high variability between protocols: (1) the types of antibodies used, (2) the usage of autofluorescence-reducing agents, (3) antigen retrieval methods, and (4) permeabilization. Therefore, in vitro immunofluorescence staining protocols were systemically adapted and improved in this work to make them applicable for different species (mouse, human) and tissues (skin, intestine, lung, liver, heart, spinal disc). After fixation and paraffin-embedding, 3 &#181;m thick sections were mounted onto slides. These samples were stained with primary antibodies for myeloperoxidase (MPO), citrullinated histone H3 (H3cit), and neutrophil elastase (NE) according to a modified staining protocol. The slides were stained with secondary antibodies and examined using a widefield fluorescence microscope. The results were analyzed according to an evaluation sheet, and differences were recorded semi-quantitatively.
Here, we present an optimized NET staining protocol suitable for different tissues. We used a novel primary antibody to stain for H3cit and reduced non-specific staining with an autofluorescence-reducing agent. Furthermore, we demonstrated that NET staining requires a constant high temperature and careful handling of samples.

저자 스포트라이트: 호중구 세포외(Neutrophil Extracellular Traps Imaging in Human and Mouse Tissue) 

Neutrophil extracellular traps (NETs) are associated with various diseases, and immunofluorescence is often used for their visualization. However, there are various staining protocols, and, in many cases, only one type of tissue is examined. Here, we establish a generally applicable protocol for staining NETs in mouse and human tissue.

인간 및 생쥐 조직에서 호중구 세포외 트랩의 면역형광 이미징

Neutrophil extracellular traps (NETs) are released by neutrophils as a response to bacterial infection or traumatic tissue damage but also play a role in ...