The authors gratefully acknowledge Drs James Roth, William Nauseef and Kayoko Kimura and Mr Tom Skadow for assistance with development of the canine neutrophil protocols. RDG is supported by a Wellcome Trust Fellowship ref: WT093767MA.

모든 실험은 아이오와 주립 대학 기관 동물 관리 및 사용위원회에서 윤리적 허가 하였다. 1. 혈액 컬렉션 <ol><li> 직접 항응고제로 복재, 두부 또는 경정맥에서 혈액의 9 ml의 그리기 (예를 들어, 에틸렌 디아민 테트라 아세트산 (EDTA), 1.8 mg의 K 2 EDTA / ml의 혈액) vacutainers, 또는 EDTA 함유 혈액 튜브에 즉시 전송 주사기로. 부드럽게 굴러 또는 혈액 항응고제의 적절한 혼합을 보장하기 위해 혈액 튜브를 반전한다. </li></ol> 2. 호중구 격리 <ol><li> 한 50㎖ 원추형 튜브에 멸균 실온 동량 혈액을 3 % 혼합하여 부드러운 반전 덱스-500 (0.9 % 멸균 식염수에 만든). 실온에서 18 ~ 20 분 동안 똑바로 서 둡니다. 그것은 더 이상 낮은 적색 층에 의해 오염없이 수집 할 수 없습니다 때까지 새로운 튜브에 밀짚 색깔의 상위 계층을 전송합니다. 일의 적혈구즉 원래 튜브는 폐기 될 수있다. </li><li> 4 ℃에서 10 분 동안 500 XG에 뜨는을 원심 분리기. 오프 그리기 및 상층 액을 버린다. 수동 10 ㎖를 실온 멸균 인산 완충 식염수 (PBS)으로 세포 펠렛을 다시 일시. 그 텍싱이 프로토콜의 모든 단계에서 다시 일시 중지 호중구에 사용하지 않아야합니다. 수동으로하지 않고 또한 세포 수율을 극대화하기 위해 프로토콜을 통해 추천 상층 액을 붓는 것보다, 오프 그리기. </li><li> 한 50㎖ 원추형 튜브에 실온 polysucrose 나트륨 diatrizoate 세포 분리 용액 (예, Histopaque 1077) 10 ㎖를 추가한다. 조심스럽게 세포 분리 용액을 통해 셀 - 함유 용액을 층을 포함한다. 셀의 수율을 감소시킬 수 실온으로 평형화되지 않았다고하여 세포 분리 용액을 참고. </li><li> 브레이크 해제와 함께 실온에서 30 분 동안 300 XG에 원심 분리기. </li><li> 호중구는 그라데이션의 가장 낮은 층에있는 바와 같이, 오프 그릴과 uppe을 폐기혈장, 혈소판, 단핵 세포의 좁은 대역 흰색 및 밀도 구배 매체를 클리어 층 상층 이루어지는 R 3 층. </li><li> 나머지 적혈구를 용해시키기 위해, 실온에서 물 10ml에 호중구 펠렛을 다시 일시. </li><li> 30 초 후, 멸균 실온 1.8 % 염화 나트륨 10 ㎖를 첨가하여 긴장성 복원하고 온화한 역전에 의해 혼합한다. </li><li> 4 ℃에서 5 분 동안 500 XG에 원심 분리기. 상층 액을 그려 버린다. </li><li> 펠렛 여전히 적색 인 경우, 용해하는 단계를 반복한다. 펠렛은 백색 (또는 용해 이미 두 번 수행 된 경우), 부드럽게 멸균 PBS 10 ㎖로 세포를 다시 정지합니다. </li><li> 자동 카운터 또는 혈구를 사용하여 세포를 계산합니다. 일반적인 금리는 혈액 1 ㎖ 당 적어도 10 6 세포이다. </li><li> 4 ℃에서 5 분 동안 500 XG에 원심 분리기. 상층 액을 그려 버린다. </li><li> 멸균 PBS 실온에서 원하는 농도로 세포를 다시 일시. FOR DNA를 분리 분석은 ml의 당 5 × 10 6 세포에서, 섹션 2에 resuspend을 설명했다. </li><li> 경우에 따라, 세포의 소량을 직접 전송하거나 유리 슬라이드에 cytocentrifuge을 사용. 세포가 건조 얼룩 (가) &#39;지침을 제조에 따라 빠른 고정 및 염색 키트를 사용 할 수 있습니다. 현미경으로 관찰 할 때 호중구의 분리가 성공한 경우 유핵 세포의 적어도 95 %는 최소한의 오염 적혈구와 과립구 (호중구, 호염기구, 호산구)이어야한다. </li></ol> 3. DNA 자료 분석 <ol><li> 호중구 차단 후 바로 DNA 자료 분석을 설정합니다. </li><li> 세포 응집 광 현미경 원액 시험에 있으면, 즉시 분석을 설정하기 전에 멸균 70 ㎛의 멸균 필터를 이용하여 세포 현탁액을 통과한다. </li><li> 멸균 96 웰 플레이트의 각 실험 웰에 5 × 104 세포 / 웰을 추가; 작용제 및 로스웰 파크 메모페놀 레드와 100-200 μL의 최종 부피로 0.5 % 열 - 불 활성화 소 태아 혈청이없는 리알 연구소-1640 (RPMI) 매체. 호중구 원액 PBS 동량으로 대체되는 작용제 당 적어도 두 블랭크 웰을 포함한다. 각 작용제에 대한 빈 우물을 설정하면 형광 분석과 함께 작용제에 의해 작용제 또는 간섭의 DNA 오염을 배제 중요합니다. </li><li> 탄산 가스 (4 %) 인큐베이터에서 30 분 동안 39 ℃에서 인큐베이션. </li><li> 원하는 농도와 세포 투과성 핵산 염료에 효능을 추가합니다. 다른 핵산 염료 사이에 다릅니다 세포 투과성 염료의 최적 농도를합니다. 작용제 미디어 동량으로 대체되는 무 자극 제어는 각각의 실험에 포함되어야한다. 주 : 웰 사이의 일관된 상태를 유지하기 위해 배양액의 유사한 양의 효능을 희석하는 것이 바람직하다. (200)의 최종 아니라 볼륨 &amp;# 181; 난 성공적으로 사용되어왔다이이 변경되는 경우, 잘 볼륨은 모든 조건이 동일 유지해야한다. 포볼 -12- 미리 스테이트 -13- 아세테이트 (PMA) (최종 농도 ≥0.1 μM) 혈소판 활성화 인자 (PAF) (최종 농도 ≥31 μM)을 양성 대조군으로 사용할 수있다. 작용제 중복 적어도 측정해야한다. </li><li> 39 ° C에서 품어. 2시간 후, 또는 소정의 간격으로 마이크로 플레이트 판독기를 사용하여 형광을 측정한다. 최적의 시간 간격을 사용 작용제에 따라 달라질 수 있습니다. 참고 : 파장이 사용 염료에 따라 달라집니다. </li><li> 평균 형광을 계산하기 전에 빈 형광을 뺍니다. </li><li> 개인간 및 플레이트 사이의 비교를 위해, 비 자극 된 웰의 평균 형광 자극 웰의 평균 형광을 나누어 형광 폴드 변화를 계산한다. </li></ol>

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저자는 공개 아무것도 없어.

제시된 DNA 분리 분석은 세포 DNA에 대한 용이 한 정량 분석한다. 이 방법은 다른 종에서 NET 형성을 평가하기 위해 사용 된 유사한 기술에서 채택되었지만, 원심 속도 작용제 농도와 항온 처리 시간은 개과 호중구 8,17,18 함께 사용하기위한 방법을 최적화하기 위해 변경되었다. 유사한 조정이 다른 종의 방법을 적용하도록 할 수있다. 분석은 유동 세포 계측법 또는 이미지 분석으로 NETosis을 ?...

만 70 애완 동물 개 1 혼자 미국에 있습니다. 소중한 가족으로,이 동물은 종종 가장자리 치료를 절단받을 수 있습니다. 그들은 우리의 환경을 공유 마찬가지로 때문에, 개는 인간의 질병 1의 병인과 치료에 대한 통찰력을 제공 할 수 있습니다. 그러나, 의학적 치료 또는 그 반대에 인간 의약 발견 변환 여부는 완전히 같은 선천성 면역 반응 심지어 고도로 보존 시스템 종 변형을 특성화하는 것이 중요하다. 송곳니와 인간의 선천성 면역 시스템 사이의 차이의 예는 개 호중구 (2) 높은 표현 CD4을 포함한다; 개 3의 세포질 플라 젤린 센서 IPAF의 기능 동족체의 부재와 육식 4에서 카스파 제 1/4 하이브리드의 표현. 호중구 세포 트랩 (그물) 선천성 면역 (5)의 비교적 최근에 발견 된 구성 요소입니다. 그물 네트워크입니다감염성 또는 염증성 자극 (6)의 넓은 범위에 응답하여 해제 DNA, 핵 단백질의 입상. NET 같은 구조 닭 7, 8 물고기, 연체 동물류 (9, 10)을 포함하는 많은 종 acoelomates 통해 입증되었지만 종 차이가있다. 예를 들어, 쥐의 호중구 인간 호중구보다 NETosis 자극에 더 느리게 응답, 덜 확산 그물 (11)을 형성한다. 그물 직접 병원균 12,13 죽이는 데 관여 할 수 이상의 논쟁 미생물 포획, 복수 종 증거하는 큰 몸체있다. 그러나 NET 구성 요소는 또한, 조직 손상을 강화 14, 15을자가 항원으로 혈전증 행동을 촉진. 그물의 유익하고 해로운 영향 사이의 균형은 종과 관심의 조건에 모두 그물을 조사하는 것이 중요하다 제안, 다른 질병과 다른 종 사이에 차이가있을 수 있습니다. 여기에 우리유도와 송곳니 호중구 그물의 방출을 측정하기위한 간단한 프로토콜을 설명한다. 이 방법은 호중구 (16)을 분리하여 다른 종 NETosis을 유도하기 위해 사용 된 것과 유사하지만, 이러한 효능 제 농도 및 배양 시간 등의 조건은 개과 호중구에 대해 최적화되었다. 유사한 NET 정량화 DNA 분리 분석은 다른 종에서 설명되었지만, 여기에 제시된 방법은 또한 개 8,17,18에 최적화되어있다.

<table border="0" cellpadding="0" cellspacing="0" width="546">
	<colgroup>
		<col />
		<col />
		<col span="2" />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:204px;">Plastic Whole Blood tube with spray-coated K2EDTA</td>
			<td style="width:215px;">BD</td>
			<td style="width:64px;">367835</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Histopaque-1077</td>
			<td>Sigma-Aldrich</td>
			<td>10771</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dextran-500</td>
			<td>Accurate chemical and scientific corp.</td>
			<td>AN228410</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphate buffered saline</td>
			<td>ThermoFisher scientific</td>
			<td>20012043</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal bovine calf serum, heat inactivated</td>
			<td>ThermoFisher scientific</td>
			<td>10100139</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RPMI Media 1640, without phenol red or L-glutamine</td>
			<td>ThermoFisher scientific</td>
			<td>32404-014</td>
			<td>Should be free from phenol red</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96 well flat bottomed sterile polystyrene plate</td>
			<td>Falcon</td>
			<td>353072</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phorbol 12-myristate 13-acetate</td>
			<td>Sigma-Aldrich</td>
			<td>P1585</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Platelet activating factor</td>
			<td>Sigma-Aldrich</td>
			<td>P4904</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SYTOX Green Nucleic Acid Stain</td>
			<td>ThermoFisher scientific</td>
			<td>S7020</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Synergy 2 Multi-Mode Reader</td>
			<td>BioTek</td>
			<td>NA</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

이 프로토콜을 이용하여, 양성 대조군, 및 PAF PMA로 호중구를 자극 후의 형광 강한 배의 변화가있을 것이다. 비 자극 세포 (범위 2.0-5.8, N = 5 개) 18에 비해 형광의 평균 4.0 배 증가에서 1 시간 결과 31 μM PAF와 그림 1B, 송곳니 호중구의 자극에 도시 된 바와 같이. 0.1 μM (도 1a)에서 PMA 2 시간까지 형광을 증가시키지 않는 느린 작용제하지만 궁극?...

Watch this Scientific Journal Video about A Simple Fluorescence Assay for Quantification of Canine Neutrophil Extracellular Trap Release at JoVE.com

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

호중구 세포 트랩 (그물) DNA, 히스톤 및 호중구 단백질의 네트워크입니다. 선천성 면역 반응의 구성 요소이지만, 그물자가 면역 및 혈전증에 연루되어있다. 이 프로토콜은 개과 호중구 분리 및 마이크로 플레이트 형광 분석법을 이용하여 그물 정량화하기위한 간단한 방법을 설명한다.

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 ...

a-simple-fluorescence-assay-for-quantification-canine-neutrophil

Research

JoVE Journal

Immunology and Infection

8.0K 조회수.  Iowa State University.  호중구 세포 트랩 (그물) DNA, 히스톤 및 호중구 단백질의 네트워크입니다. 선천성 면역 반응의 구성 요소이지만, 그물자가 면역 및 혈전증에 연루되어있다. 이 프로토콜은 개과 호중구 분리 및 마이크로 플레이트 형광 분석법을 이용하여 그물 정량화하기위한 간단한 방법을 설명한다. 

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

Neutrophil Extracellular Traps: How to Generate and Visualize Them

Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them intracellularly when their antimicrobial granules fuse with the phagosome. We found that neutrophils have an additional way of killing microorganisms: upon activation, they release granule proteins and chromatin that together form extracellular fibers that bind pathogens. These novel structures, or Neutrophil Extracellular Traps (NETs), degrade virulence factors and kill bacteria1, fungi2 and parasites3. The structural backbone of NETs is DNA, and they are quickly degraded in the presence of DNases. Thus, bacteria expressing DNases are more virulent4. Using correlative microscopy combining TEM, SEM, immunofluorescence and live cell imaging techniques, we could show that upon stimulation, the nuclei of neutrophils lose their shape and the eu- and heterochromatin homogenize. Later, the nuclear envelope and the granule membranes disintegrate allowing the mixing of NET components. Finally, the NETs are released as the cell membrane breaks. This cell death program (NETosis) is distinct from apoptosis and necrosis and depends on the generation of Reactive Oxygen Species by NADPH oxidase5. 
Neutrophil extracellular traps are abundant at sites of acute inflammation. NETs appear to be a form of innate immune response that bind microorganisms, prevent them from spreading, and ensure a high local concentration of antimicrobial agents to degrade virulence factors and kill pathogens thus allowing neutrophils to fulfill their antimicrobial function even beyond their life span. There is increasing evidence, however, that NETs are also involved in diseases that range from auto-immune syndromes to infertility6.
We describe methods to isolate Neutrophil Granulocytes from peripheral human blood7 and stimulate them to form NETs. Also we include protocols to visualize the NETs in light and electron microscopy.

Neutrophil Extracellular Traps (NETs) are an important innate immune mechanism to fight pathogenic bacteria, fungi and parasites. Here we describe methods to isolate neutrophil granulocytes from human blood and to activate them to form NETs. We present preparation techniques to visualize NETs in light and electron microscopy.

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 ...

연구

면역학 및 감염병학

Concurrent Quantification of Cellular and Extracellular Components of Biofilms

Confocal laser scanning microscopy (CLSM) is a powerful tool for investigation of biofilms. Very few investigations have successfully quantified concurrent distribution of more than two components within biofilms because: 1)&#160;selection of fluorescent dyes having minimal spectral overlap is complicated, and 2)&#160;quantification of multiple fluorochromes poses a multifactorial problem. Objectives: Report a methodology to quantify and compare concurrent 3-dimensional distributions of three cellular/extracellular components of biofilms grown on relevant substrates. Methods: The method consists of distinct, interconnected steps involving biofilm growth, staining, CLSM imaging, biofilm structural analysis and visualization, and statistical analysis of structural parameters. Biofilms of Streptococcus mutans (strain UA159) were grown for 48 hr on sterile specimens of Point 4 and TPH3 resin composites. Specimens were subsequently immersed for 60 sec in either Biot&#232;ne PBF (BIO) or Listerine Total Care (LTO) mouthwashes, or water (control group; n=5/group). Biofilms were stained with fluorochromes for extracellular polymeric substances, proteins and nucleic acids before imaging with CLSM. Biofilm structural parameters calculated using ISA3D image analysis software were biovolume and mean biofilm thickness. Mixed models statistical analyses compared structural parameters between mouthwash and control groups (SAS software; &#945;=0.05). Volocity software permitted visualization of 3D distributions of overlaid biofilm components (fluorochromes). Results: Mouthwash BIO produced biofilm structures that differed significantly from the control (p&#60;0.05) on both resin composites, whereas LTO did not produce differences (p&#62;0.05) on either product. Conclusions: This methodology efficiently and successfully quantified and compared concurrent 3D distributions of three major components within S. mutans biofilms on relevant substrates, thus overcoming two challenges to simultaneous assessment of biofilm components. This method can also be used to determine the efficacy of antibacterial/antifouling agents against multiple biofilm components, as shown using mouthwashes. Furthermore, this method has broad application because it facilitates comparison of 3D structures/architecture of biofilms in a variety of disciplines.

A protocol for the concurrent quantification and comparison of three cellular and extracellular components within biofilms is presented. The methodology involves the use of confocal laser scanning microscopy, biofilm structural analysis and visualization software, and statistical analysis software.

Confocal laser scanning microscopy (CLSM) is a powerful tool for investigation of biofilms. Very few investigations have successfully quantified ...

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils.&#160;This process has the potential to be destructive to tissues if excessive or held in an unresolved state.&#160; Cocultured in vitro models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration.&#160;This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil.&#160;Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface.&#160;The in vitro model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa (PAO1).&#160;Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli (MC1000).&#160; The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls.&#160;This in vitro model system can be manipulated and applied to other mucosal surfaces.&#160;Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections.&#160;A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases.

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Neutrophil trans-epithelial migration in response to mucosal bacterial infection contributes to epithelial injury and clinical disease.&#160;An in vitro model has been developed that combines pathogen, human neutrophils, and polarized human epithelial cell layers grown on transwell filters to facilitate investigations towards unraveling the molecular mechanisms orchestrating this phenomenon.

시험관 내 병원균 유도 호중구 트랜스 상피 이동을 평가하는 공동 문화 분석

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to ...

Human Neutrophil Flow Chamber Adhesion Assay

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion involves many different adhesion molecules including members of the &beta;2 integrin family and their counter-receptors of the ICAM family. Recently, naturally occurring genetic variants in both &beta;2 integrins and ICAMs are reported to be associated with autoimmune disease. Thus, the quantitative adhesive capacity of neutrophils from individuals with varying allelic forms of these adhesion molecules is important to study in relation to mechanisms underlying development of autoimmunity. Adhesion studies in flow chamber systems can create an environment with fluid shear stress similar to that observed in the blood vessel environment in vivo. Here, we present a method using a flow chamber assay system to study the quantitative adhesive properties of human peripheral blood neutrophils to human umbilical vein endothelial cell (HUVEC) and to purified ligand substrates. With this method, the neutrophil adhesive capacities from donors with different allelic variants in adhesion receptors can be assessed and compared. This method can also be modified to assess adhesion of other primary cell types or cell lines.

A method of quantitating neutrophil adhesion is reported. This method creates a dynamic flow environment similar to that encountered in a blood vessel. It allows the investigation of neutrophil adhesion to either purified adhesion molecules (ligand) or endothelial cell substrate (HUVEC) in a context similar to the in vivo environment with sheer stress.

인간 호중구 유량 용기 접착 분석

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion ...

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 ...

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 ...

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling and cellular processes that control efferocytosis. This quantification can be difficult to perform as apoptotic cells are often efferocytosed piecemeal, thus necessitating methods which can accurately delineate between the efferocytosed portion of an apoptotic target versus residual unengulfed cellular fragments. The approach outlined herein utilizes dual-labeling approaches to accurately quantify the dynamics of efferocytosis and efferocytic capacity of efferocytes such as macrophages. The cytosol of the apoptotic cell is labeled with a cell-tracking dye to enable monitoring of all apoptotic cell-derived materials, while surface biotinylation of the apoptotic cell allows for differentiation between internalized and non-internalized apoptotic cell fractions. The efferocytic capacity of efferocytes is determined by taking fluorescent images of live or fixed cells and quantifying the amount of bound versus internalized targets, as differentiated by streptavidin staining. This approach offers several advantages over methods such as flow cytometry, namely the accurate delineation of non-efferocytosed versus efferocytosed apoptotic cell fractions, the ability to measure efferocytic dynamics by live-cell microscopy, and the capacity to perform studies of cellular signaling in cells expressing fluorescently-labeled transgenes. Combined, the methods outlined in this protocol serve as the basis for a flexible experimental approach that can be used to accurately quantify efferocytic activity and interrogate cellular signaling pathways active during efferocytosis.

Efferocytosis, the phagocytic removal of apoptotic cells, is required to maintain homeostasis and is facilitated by receptors and signaling pathways that allow for the recognition, engulfment, and internalization of apoptotic cells. Herein, we present a fluorescence microscopy protocol for the quantification of efferocytosis and the activity of efferocytic signaling pathways.

단일 세포 형광 현미경 검사 법에 의해 Efferocytosis의 정량화

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...

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. ...

In Vitro Stimulation and Visualization of Extracellular Trap Release in Differentiated Human Monocyte-derived Macrophages

The release of extracellular traps (ETs) by neutrophils has been identified as a contributing factor to the development of diseases related to chronic inflammation. Neutrophil ETs (NETs) consist of a mesh of DNA, histone proteins, and various granule proteins (i.e., myeloperoxidase, elastase, and cathepsin G). Other immune cells, including macrophages, can also produce ETs; however, to what extent this occurs in vivo and whether macrophage extracellular traps (METs) play a role in pathological mechanisms has not been examined in detail. To better understand the role of METs in inflammatory pathologies, a protocol was developed for visualizing MET release from primary human macrophages in vitro, which can also be exploited in immunofluorescence experiments. This allows further characterization of these structures and their comparison to ETs released from neutrophils. Human monocyte-derived macrophages (HMDM) produce METs upon exposure to different inflammatory stimuli following differentiation to the M1 pro-inflammatory phenotype. The release of METs can be visualized by microscopy using a green fluorescent nucleic acid stain that is impermeant to live cells (e.g., SYTOX green). Use of freshly isolated primary macrophages, such as HMDM, is advantageous in modeling in vivo inflammatory events that are relevant to potential clinical applications. This protocol can also be used to study MET release from human monocyte cell lines (e.g., THP-1) following differentiation into macrophages with phorbol myristate acetate or other macrophage cell lines (e.g., the murine macrophage-like J774A.1 cells).

Presented here is a protocol to detect macrophage extracellular trap (MET) production in live cell culture using microscopy and fluorescence staining. This protocol can be further extended to examine specific MET protein markers by immunofluorescence staining.

분화 한 인간 단핵구 유래 대식세포에서 세포 외 트랩 방출의 시험관 내 자극 및 시각화

The release of extracellular traps (ETs) by neutrophils has been identified as a contributing factor to the development of diseases related to chronic ...

개 호중구 세포 외 트랩 릴리스의 정량화를위한 ​​간단한 형광 분석

개 호중구 세포 외 트랩 릴리스의 정량화를위한 간단한 형광 분석