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.

佐治亚大学的机构审查委员会批准的人类主体研究，以收集来自健康志愿者（UGA＃2012-10769-06）外周血。5,7,8志愿者签署抽血前所需的知情同意书。在这篇文章中进行的研究符合涉及赫尔辛基宣言人体医学研究的伦理准则。 1.人外周血中性粒细胞的分离（图1） 注意:有几种方法分离自外周血的中性粒细胞。以下方案提供了一种可能性。该协议产生大量能够对外界刺激释放教师的非激活的人中性粒细胞。使用40毫升由静脉穿刺健康志愿者获得的全血。7,9 <ol><li>分装20毫升血液进入两个50毫升锥形管。加入10 mL 6％右旋糖酐。轻轻混匀。 </li><li>经过20分钟转移白细胞RICħ上层相而不采取任何沉淀的红细胞成清洁50ml锥形管中，并用无菌PBS填满它。 </li><li>离心机在400 xg离心10分钟。在4毫升重悬沉淀无菌PBS。 </li><li>制备85％的5个步骤的Percoll梯度-80％-75％-70％-65％在两个15毫升锥形管和层2毫升白细胞悬浮液在每个梯度的顶端。 </li><li>离心机800 XG与刹车关闭30分钟。 </li><li>收集在75％/ 80％的所有细胞（中性粒细胞）在所述层和70％/ 75％的界面。 </li><li>在PBS中洗涤细胞两次（350 XG，10分钟，RT）和使用血球计数细胞。 </li></ol> 2.使用微孔板荧光计外DNA释放的动力学测量注意:此方法能够在不可透过膜的DNA结合在96孔微孔板格式指示的DNA释放的染料（ 图2）的荧光变化的测量。 <ol><li>制备悬浮液在测定培养基嗜中性粒细胞（HBSS +1％（V / V）的自体血清+ 5mM葡萄糖）以2×10 6个细胞/ ml的浓度。 </li><li>添加5微摩尔/升的膜不可渗透的DNA结合染料。轻轻混匀。 </li><li>等分试样的人类嗜中性粒细胞（50μl/孔）到96孔黑色，透明底微板。确保测定培养基覆盖井的整个底部。如果没有，轻轻敲击侧面的板。 </li><li>预热10分钟的含板中性白细胞在37℃。 </li><li>在此期间，在它们的最终浓度的两倍制备刺激方案中使用（在37℃的温水测定培养基）。作为阳性NET控制使用人嗜中性粒用100nM佛波醇肉豆蔻乙酸酯（PMA）4小时触发最大净释放。-5,8- PMA刺激4小时，确保最大净释放而自发NET形成仍然很低5,8。 </li><li>制备微孔板荧光计进行测定（4小时，37℃，激发530nm处和用于发射590纳米）。&lt;/ LI&gt; <li>通过加入50μl的刺激溶液至50μl嗜中性粒细胞悬浮液刺激嗜中性粒细胞。使用0.5微克/毫升皂素于一体的测量最大DNA释放信号。 </li><li>将板阅读器，并测量每2分钟没有晃动的荧光变化4小时。 </li><li>进行数据分析，计算随时间的荧光归增加。 <ol><li>从端点值减去基线荧光所有井包括皂苷（ 图2A，B）。上升皂素荧光应该是最高的信号。它被称为"最大的DNA释放"（ 图2A）。 </li><li>由最大DNA释放除以未知样品的荧光增大计算未知样品中的DNA释放百分比。 </li><li>重复的平均结果之和作为"最大的DNA释放％"（ 图2C）。 </li></ol></li></ol> 3. NET表单的定量通过通货膨胀MPO-DNA和HNE-DNA化验ELISA 注意:在我们的实验室这些测定改性（MPO-DNA）或建立（HNE-DNA）通过测量MPO-DNA和HNE-DNA复合物的水平的定量NET形成5。 <ol><li>涂层的96孔高结合过夜捕捉抗体（50μl/孔）的能力的ELISA平板:抗MPO（1:2,000稀释度）或抗HNE（1:2,000）。 </li><li>洗涤ELISA板三次，用PBS（200μl/孔）并在室温用5％BSA和0.1％人血清白蛋白阻断它们为2小时（200μl/孔）。用PBS洗涤三次。 </li><li>在96孔微孔板以100,000个细胞/孔在100μl测定培养基的密度种子嗜中性粒细胞，并刺激网的形成。孵育细胞:4小时37℃。 </li><li>加入2 U / ml的DNA酶中性粒细胞上清液进行有限的DNA酶消化，以及混合。让他们在室温。 </li><li>通过加入11微升25mM的EGTA（最终浓度2.5毫摩尔）停止15分钟后的DNA酶。拌匀。苏收集pernatants干净的离心管。自旋样品在300 xg离心除掉细胞碎片在RT 5分钟。转让其上清液到干净的离心管。让他们在冰，直到准备。 </li><li>使用预先制备的"NET标准"的等分试样。 注意:NET-标准是由至少5个不同的，独立人供体获得的PMA刺激嗜中性粒细胞的DNA酶消化的上清液的混合。 <ol><li>要使用相同的标准很长一段时间，收集并从每个捐赠者分装大量中性粒细胞上清。收集例如2毫升PMA刺激嗜中性粒细胞的DNA酶消化的上清液将导致200等份（各10微升）足以为200 ELISA板。数据表征NET标准在图5中。 </li><li>刺激人中性粒细胞用100nM PMA 4小时，并应用DNA酶消化，以根据步骤3.4-3.5其上清液。 </li><li>收集，游泳池，分装（10微升）和自由泽（-20°C），中性粒细胞上清液。 </li><li>为了准备"网标"，从解冻至少五个独立的供体获得一个等份/供体，将它们混合，并保持他们在冰上。 </li><li>丢弃解冻的样品，并使用新鲜的下一个实验。 </li><li>准备1:PBS + EGTA网标准的2系列稀释。 </li></ol></li><li>稀释DNase1消化的样品（标准和未知上清液）20倍的PBS + EGTA和分装它们涂有捕获抗体ELISA板。 </li><li>孵育过夜ELISA板在4℃下，并用PBS洗三次。 </li><li>应用检测抗体溶液（100μl/孔）:辣根过氧化物酶缀合的抗DNA抗体（1:500，鼠标）。孵育在黑暗1小时。 </li><li>洗涤四次后，用PBS加TMB过氧化物酶底物:100微升/孔，30分钟。蓝颜色是过氧化物酶活性（母语存在）的指示。 </li><li>通过加入100μl/孔的1M HCl终止反应。该蓝色的解决方案会变成黄色（ 图5A）。 </li><li>在使用微板光度计450nm处读取吸光度。 </li><li>进行数据分析，计算相对于NET标准的MPO-DNA或HNE-DNA复合物的量。 <ol><li>减去从所有样品的测定培养基的背景光密度值。 </li><li>绘制针对其相对净含量（Y轴）（ 图5A）将稀释的标准样品的吸光度值（X轴）。 </li><li>使用该标准曲线的非饱和范围建立趋势线（采用最佳指数拟合从所使用的软件）（ 图5A）。这条趋势线的方程式决定OD值转化为定量母语的金额。 </li><li>插入未知数的测量OD值到从3.13.3趋势线方程，这将使未知样品为NET-标准（ 图5A）的百分比在教师的量。 </li><li>计算重复的（一式三份）平均值，并提交最终的数据（ 图5C）"MPO-DNA或HNE-DNA复合物（标准的百分比）的量。" </li></ol></li></ol>

<ol>
	<li>Brinkmann, V., et al. <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 (2004).</li><li>Fuchs, T. A., et al. <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>Kessenbrock, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Netting+neutrophils+in+autoimmune+small-vessel+vasculitis.">Netting neutrophils in autoimmune small-vessel vasculitis.</a> Nat Med. 15, 623-625 (2009).</li><li>Leffler, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Degradation+of+neutrophil+extracellular+traps+co-varies+with+disease+activity+in+patients+with+systemic+lupus+erythematosus.">Degradation of neutrophil extracellular traps co-varies with disease activity in patients with systemic lupus erythematosus.</a> Arthritis Res Ther. 15, R34 (2013).</li><li>Yoo, D. G., Floyd, M., Winn, M., Moskowitz, S. M., Rada, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NET+formation+induced+by+Pseudomonas+aeruginosa+cystic+fibrosis+isolates+measured+as+release+of+myeloperoxidase-DNA+and+neutrophil+elastase-DNA+complexes.">NET formation induced by Pseudomonas aeruginosa cystic fibrosis isolates measured as release of myeloperoxidase-DNA and neutrophil elastase-DNA complexes.</a> Immunol Lett. 160, 186-194 (2014).</li><li>Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+hypercitrullination+mediates+chromatin+decondensation+and+neutrophil+extracellular+trap+formation.">Histone hypercitrullination mediates chromatin decondensation and neutrophil extracellular trap formation.</a> J Cell Biol. 184, 205-213 (2009).</li><li>Pang, L., Hayes, C. P., Buac, K., Yoo, D. G., Rada, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudogout-associated+inflammatory+calcium+pyrophosphate+dihydrate+microcrystals+induce+formation+of+neutrophil+extracellular+traps.">Pseudogout-associated inflammatory calcium pyrophosphate dihydrate microcrystals induce formation of neutrophil extracellular traps.</a> J Immunol. 190, 6488-6500 (2013).</li><li>Yoo, D. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Release+of+cystic+fibrosis+airway+inflammatory+markers+from+Pseudomonas+aeruginosa-stimulated+human+neutrophils+involves+NADPH+oxidase-dependent+extracellular+DNA+trap+formation.">Release of cystic fibrosis airway inflammatory markers from Pseudomonas aeruginosa-stimulated human neutrophils involves NADPH oxidase-dependent extracellular DNA trap formation.</a> J. Immunol. 192, 4728-4738 (2014).</li><li>Brinkmann, V., Laube, B., Abu Abed, ., Goosmann, U., Zychlinsky, C., 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,+how+to+generate+and+visualize+them.">Neutrophil extracellular traps, how to generate and visualize them.</a> J. Vis. Exp. , (2010).</li><li>Rada, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyocyanin-enhanced+neutrophil+extracellular+trap+formation+requires+the+NADPH+oxidase.">Pyocyanin-enhanced neutrophil extracellular trap formation requires the NADPH oxidase.</a> PLoS One. 8, e54205 (2013).</li><li>Neumann, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+antimicrobial+peptide+LL-37+facilitates+the+formation+of+neutrophil+extracellular+traps.">The antimicrobial peptide LL-37 facilitates the formation of neutrophil extracellular traps.</a> Biochem J. 464, 3-11 (2014).</li><li>Leon, S. A., Shapiro, B., Sklaroff, D. M., Yaros, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free+DNA+in+the+serum+of+cancer+patients+and+the+effect+of+therapy.">Free DNA in the serum of cancer patients and the effect of therapy.</a> Cancer Res. 37, 646-650 (1977).</li><li>Raptis, L., Menard, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+and+characterization+of+plasma+DNA+in+normals+and+patients+with+systemic+lupus+erythematosus.">Quantitation and characterization of plasma DNA in normals and patients with systemic lupus erythematosus.</a> J Clin Invest. 66, 1391-1399 (1980).</li><li>Chang, C. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elevated+cell-free+serum+DNA+detected+in+patients+with+myocardial+infarction.">Elevated cell-free serum DNA detected in patients with myocardial infarction.</a> Clin Chim Acta. 327, 95-101 (2003).</li></ol>

The authors have nothing to disclose.

英语教师代表是中性粒细胞杀死病原体。1虽然教师的文学过去十年里，因为他们发现，与他们的生物学，机制和调控作用，一些重要的问题仍不清楚不断扩大在一个迷人的新机制。适当的方法已被开发来衡量英语教师，这非常独特的抗菌机理。本文介绍了可用于定量英语教师在高通量的方式方法。第一测定如下的膜不透DNA结合染料的荧光的动力学。此法提供了大量的斜坡上，距离中性粒?...

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.

在这个手稿附图描述嗜中性粒细胞分离，实验程序和数据分析的解释本代表性结果的方法。 图1示出人中性粒细胞的制备的顺序步骤。该协议仅表示嗜中性粒细胞分离的可能的方式。它会产生大量的休息能够刺激后释放的外籍英语教师的中性粒细胞。 图2显示了基于荧光的DNA释放试验是如何工作的。使用DNA释放的荧光酶标仪动力学在人嗜中性粒细?...

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.

胞外DNA释放的高通量测量和人中性粒定量NET形成<em&gt;体外</em

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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Immunology and Infection

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

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

视频: 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).

使用双光子显微镜在受感染的肺部的嗜中性粒细胞吞噬和NET形成的直接观察

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

科研

免疫与感染

Quantitative Measurement of the Immune Response and Sleep in Drosophila

A complex interaction between the immune response and host behavior has been described in a wide range of species. Excess sleep, in particular, is known to occur as a response to infection in mammals 1 and has also recently been described in Drosophila melanogaster2. It is generally accepted that sleep is beneficial to the host during an infection and that it is important for the maintenance of a robust immune system3,4. However, experimental evidence that supports this hypothesis is limited4, and the function of excess sleep during an immune response remains unclear. We have used a multidisciplinary approach to address this complex problem, and have conducted studies in the simple genetic model system, the fruitfly Drosophila melanogaster. We use a standard assay for measuring locomotor behavior and sleep in flies, and demonstrate how this assay is used to measure behavior in flies infected with a pathogenic strain of bacteria. This assay is also useful for monitoring the duration of survival in individual flies during an infection. Additional measures of immune function include the ability of flies to clear an infection and the activation of NF&kappa;B, a key transcription factor that is central to the innate immune response in Drosophila. Both survival outcome and bacterial clearance during infection together are indicators of resistance and tolerance to infection. Resistance refers to the ability of flies to clear an infection, while tolerance is defined as the ability of the host to limit damage from an infection and thereby survive despite high levels of pathogen within the system5. Real-time monitoring of NF&kappa;B activity during infection provides insight into a molecular mechanism of survival during infection. The use of Drosophila in these straightforward assays facilitates the genetic and molecular analyses of sleep and the immune response and how these two complex systems are reciprocally influenced.

Quantitative Measurement of the Immune Response and Sleep in Drosophila

To understand a link between the immune response and behavior, we describe a method to measure locomotor behavior in Drosophila during bacterial infection as well as the ability of flies to mount an immune response by monitoring survival, bacterial load, and real-time activity of a key regulator of innate immunity, NF&kappa;B.

定量测定的免疫反应和睡眠模式<em&gt;果蝇</em

A complex interaction between the immune response and  host behavior has been described in a wide range of species. Excess sleep, in  particular, is known ...

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5.
Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8.
Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.

We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

定量高通量单细胞的细胞毒性T细胞的测定

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

建立一个<em&gt;在体外</em&gt;系统来研究细胞内的行为<em&gt;光滑念珠菌</em&gt;在人THP-1巨噬细胞

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

量<em在体外</em&gt;激活的主人微血管内皮细胞在静态条件下测量的检测，以中性粒细胞粘附

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

High-throughput Gene Tagging in Trypanosoma brucei

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins can give insights into their function by determining their localization within the cell and enabling interaction partner identification. We recently published a fast and scalable method to generate Trypanosoma brucei cell lines that express a tagged protein from the endogenous locus. The method was based on a plasmid we generated that, when coupled with long primer PCR, can be used to modify a gene to encode a protein tagged at either terminus. This allows the tagging of dozens of trypanosome proteins in parallel, facilitating the large-scale validation of candidate genes of interest. This system can be used to tag proteins for localization (using a fluorescent protein, epitope tag or electron microscopy tag) or biochemistry (using tags for purification, such as the TAP (tandem affinity purification) tag). Here, we describe a protocol to perform the long primer PCR and the electroporation in 96-well plates, with the recovery and selection of transgenic trypanosomes occurring in 24-well plates. With this workflow, hundreds of proteins can be tagged in parallel; this is an order of magnitude improvement to our previous protocol and genome scale tagging is now possible.

High-throughput Gene Tagging in Trypanosoma brucei

Addition of a tag to a protein is a powerful way of gaining insight into its function. Here, we describe a protocol to endogenously tag hundreds of Trypanosoma brucei proteins in parallel such that genome scale tagging is achievable.

高通量基因定位在<em&gt;布氏锥虫</em

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins ...

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.

方法来研究中性粒细胞脂质的改变和中性粒细胞胞外陷阱随后形成

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

Cell-free Biochemical Fluorometric Enzymatic Assay for High-throughput Measurement of Lipid Peroxidation in High Density Lipoprotein

Low high-density lipoprotein cholesterol (HDL-C) levels are one of the most powerful independent negative predictors of atherosclerotic cardiovascular disease (CVD). The structure and function of HDL rather than HDL-C may more accurately predict atherosclerosis. Several HDL protein and lipid compositional changes that impair HDL function occur in inflammatory states such as atherosclerosis. HDL function is usually determined by cell based assays such as cholesterol efflux assay but these assays have numerous drawbacks lack of standardization. Cell-free assays may give more robust measures of HDL function compared to cell-based assays. HDL oxidation impairs HDL function. HDL has a major role in lipid peroxide transport and high amount of lipid peroxides is related to abnormal HDL function. Lipid-probe interactions should be considered when interpreting the results of non-enzymatic fluorescence assays for measuring the lipid oxidative state. This motivated us to develop a cell-free biochemical enzymatic method to assess HDL lipid peroxide content (HDLox) that contributes to HDL dysfunction. This method is based on the enzyme horseradish peroxidase (HRP) and the fluorochrome Amplex Red that can quantify (without cholesterol oxidase) the lipid peroxide content per mg of HDL-C. Here a protocol is describedfor determination of HDL-lipid peroxidation using the fluorochrome reagent. Assay variability can be reduced by strict standardization of experimental conditions. Higher HDLox values are associated with reduced HDL antioxidant function. The readout of this assay is associated with readouts of validated cell-based assays, surrogate measures of cardiovascular disease, systemic inflammation, immune dysfunction, and associated cardiovascular and metabolic risk phenotypes. This technical approach is a robust method to assess HDL function in human disease where systemic inflammation, oxidative stress and oxidized lipids have a key role (such as atherosclerosis).

We describe here a fluorometric cell-free biochemical assay for determination of HDL-lipid peroxidation. This rapid and reproducible assay can be used to determine HDL function in large scale studies and can contribute to our understanding of HDL function in human disease.

无细胞生化荧光酶法测定高密度脂蛋白脂质过氧化的高通量测定

Low high-density lipoprotein cholesterol (HDL-C) levels are one of the most powerful independent negative predictors of atherosclerotic cardiovascular ...

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.

体外犬中性粒细胞胞外陷阱的形成: 荧光显微镜动态定量分析

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

High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In response to these damages, complex molecular machineries rapidly reseal the plasma membrane to restore its barrier function and maintain cell survival. Despite 60 years of research in this field, we still lack a thorough understanding of the cell resealing machinery. With the goal of identifying cellular components that control plasma membrane resealing or drugs that can improve resealing, we have developed a fluorescence-based high-throughput assay that measures the plasma membrane resealing efficiency in mammalian cells cultured in microplates. As a model system for plasma membrane damage, cells are exposed to the bacterial pore-forming toxin listeriolysin O (LLO), which forms large 30-50 nm diameter proteinaceous pores in cholesterol-containing membranes. The use of a temperature-controlled multi-mode microplate reader allows for rapid and sensitive spectrofluorometric measurements in combination with brightfield and fluorescence microscopy imaging of living cells. Kinetic analysis of the fluorescence intensity emitted by a membrane impermeant nucleic acid-binding fluorochrome reflects the extent of membrane wounding and resealing at the cell population level, allowing for the calculation of the cell resealing efficiency. Fluorescence microscopy imaging allows for the enumeration of cells, which constitutively express a fluorescent chimera of the nuclear protein histone 2B, in each well of the microplate to account for potential variations in their number and allows for eventual identification of distinct cell populations. This high-throughput assay is a powerful tool expected to expand our understanding of membrane repair mechanisms via screening for host genes or exogenously added compounds that control plasma membrane resealing.

Here we describe a high-throughput fluorescence-based assay that measures the plasma membrane resealing efficiency through fluorometric and imaging analyses in living cells. This assay can be used for screening drugs or target genes that regulate plasma membrane resealing in mammalian cells.

哺乳动物细胞血浆膜检测效率的高通量测量

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In ...

胞外DNA释放的高通量测量和人中性粒定量NET形成

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此视频中的章节

使用双光子显微镜在受感染的肺部的嗜中性粒细胞吞噬和NET形成的直接观察

定量测定的免疫反应和睡眠模式

定量高通量单细胞的细胞毒性T细胞的测定

建立一个

量

高通量基因定位在

方法来研究中性粒细胞脂质的改变和中性粒细胞胞外陷阱随后形成

无细胞生化荧光酶法测定高密度脂蛋白脂质过氧化的高通量测定

体外犬中性粒细胞胞外陷阱的形成: 荧光显微镜动态定量分析

哺乳动物细胞血浆膜检测效率的高通量测量