Name: fast and specific assessment of the halogenating peroxidase activity in leukocyte-enriched blood samples
Uploaded: 2016-07-28T15:00:00.000+00:00
Duration: 5 min 17 s
Description: Watch this Scientific Journal Video about Fast and Specific Assessment of the Halogenating Peroxidase Activity in Leukocyte-enriched Blood Samples at JoVE.com

This work was made possible by funding from the German Federal Ministry of Education and Research (BMBF, 1315883) as well as by the S&#228;chsische Aufbaubank (SAB) project 100116526 from a funding of the European Regional Development Fund (ERDF).

从健康志愿者获得了人类所有的血液样本，并应用白细胞浓缩协议遵循莱比锡大学医学院伦理委员会的指导方针。与大鼠血液实验是由负责当地的伦理委员会（Landesdirektion萨克森，Referat 24），根据动物护理和使用指南德国批准。 1.实验装置注意:作为用于从血液样品的红细胞的枯竭的低渗裂解过程是时间关键的步骤中，该协议的这部分预先准备所有必要的设备（ 例如，缓冲液）。 <ol><li>标号为低渗裂解1个15毫升离心管中，并为每个血液样品随后氨基苯基荧光素（APF）染色1的1.5ml样品管中。 <ol><li>如果白细胞富集将在无菌条件下进行，气流下执行此标记框。 </li></ol></li><li>制备每个样品约12mL磷酸盐缓冲盐水（PBS，10毫摩尔）。根据是否白细胞应当在无菌条件下进行分离与否，通过使用从一个供应商无菌准备溶液或通过在蒸馏水中溶解的PBS片剂或制备PBS中。检测pH值，如果必要，通过使用少量的0.1M HCl以及NaOH溶液调节pH至7.4。 <ol><li>溶解的PBS片剂（参见材料的表）在200ml蒸馏水中，得到充分的一个非无菌缓冲溶液中约16个样本。如果需要的话，灭菌通过无菌过滤该溶液。 </li></ol></li><li>制备补充有钙约1ml Hanks平衡盐溶液（HBSS）2+每个样品。 <ol><li>溶解970毫克在100毫升HBSS盐（最终体积）双蒸水。填充到最终体积前，检查pH值调整到pH为7.4。由于其较低的缓冲能力，每天一个新鲜准备这个缓冲区d通过使用少量的0.1M HCl以及NaOH溶液进行pH调节特别小心。 注:无菌溶液也可以使用，这取决于实验。该溶液可通过无菌过滤进行灭菌。 </li></ol></li><li>除了 ​​两个缓冲器，标签，并准备一管（ 例如50毫升离心管）或烧瓶（ 如250毫升量杯）预先用双蒸水的低渗裂解过程。准备每个样品约10毫升的水。 </li><li>准备与用于APF染色（第3部分），这是在冰上进行碎冰的容器。 </li><li>准备APF的等分提前，使染色过程中使用的工作方案快速制备（步骤3.2），并避免重复冻结和APF溶液中解冻。 注:APF是作为5毫克/毫升（11.81毫摩尔）储备溶液在乙酸甲酯中通常得到的。 <ol><li>冻结10-100微升的等分试样在0.5ml管中。对于APF染色最后为10μM染料浓度被使用（步骤3.8）。 </li></ol></li></ol> 2.红细胞的低渗裂解注:一个层流长椅下执行低渗裂解步骤（除了离心步骤），以避免污染。在室温下执行整个程序。作为低渗裂解是时间关键的工艺，调整水（2ml）和PBS（5毫升）加入移液器提前和启动过程之前，打开水和PBS烧瓶中。存储PBS溶液和蒸馏水在室温下在使用前。 <ol><li>由各血液试样转移100μl到适当15毫升离心管中。 注:在我们的实验血样通常含有以避免凝结10U / ml的肝素。然而，由于样品材料的不同来源的抗凝血剂的性质和浓度可能不同。其对APF染色影响应该由COMPAR测试ING结果从补充有其它类型或抗凝血剂的浓度的血液样品中获得的结果。 </li><li>加2ml蒸馏水，并使用涡旋混合器设置为中等水平混合样品。孵育样品60秒，然后加入5毫升的PBS每个样品，并使用涡旋混合器混合。 注:最多约5个样本可以并行制备。保持样品顺序相同的添加水和PBS中达到相当的温育时间。 </li><li>由PBS加成（步骤2.2）通过离心，在室温下6分钟，450×g下沉淀在所有样品中剩余的完整细胞等渗条件再生后。 </li><li>通过倾入表垃圾箱中取出上清。颠倒离心管和攻丝开到纸巾中取出尽可能多的液体越好。确保该细胞沉淀是尽可能干燥。 </li><li>像以前一样（步骤2.2）中描述重复低渗裂解过程。 ñOTE:原则上，这低渗裂解过程（步骤2.3-2.5）可以重复一次以上，这取决于混合的白细胞部分的纯度来获得。经过两次裂解步骤中所得到的混合的细胞级分剩余红细胞的份额约为25％。 </li><li>沉淀剩余的完整细胞离心，在室温下6分钟，450×g下。除去上清液（见步骤2.4）。加入500微升HBSS向沉淀并直到获得均匀的溶液轻轻地溶解。每个样品转移到相应标记的1.5ml样品管中。 </li><li>根据实验直接分析获得的样品（ 例如，通过流式细胞仪）25，染色，用荧光标记的抗体（单细胞类型的标识）32，与细胞刺激孵育（细胞活化实验）33或在冰上保存，并使用后来。根据这项研究的目的，立即执行对随后的实验由于粒混合白细胞部分有大约20小时的一个相当短的半衰期。 注意:这也适用于下面描述的过氧化物酶的活性染色。 </li></ol> 3.卤化过氧化物活性染色注:HOCl-和次溴酸生产由血液衍生血红素过氧化物酶MPO和EPO是通过使用APF，这是由称为次卤酸氧化成荧光定量。因此，如果用荧光标记抗体细胞标记与APF染色组合进行，避免用荧光素的发射信号干扰的荧光。 <ol><li>在冰上4℃下进行APF染色。 </li><li>为了制备有源滤波器的工作溶液解冻染料的适当等分试样（ 例如，10微升，11.81毫摩尔）并用HBSS稀释至恰好为1mM（ 例如，添加108.1微升缓冲到10微升）。 <ol><li>对于每个样品（500微升细胞溶解液）准备5μ微升描述APF工作液。因此，使用APF（11.81毫摩尔），这将产生118.1微升APF工作溶液（1毫摩尔），以制备约20个样品中的一个10微升等分试样。由于移液过程中的损失准备比计算值多10％APF工作液。 </li></ol></li><li>为了检查APF染色的过氧化物酶的特异性，用血红素过氧化物酶抑制剂4-氨基苯甲酸酰肼（4- ABAH）35制备对照样品。 <ol><li>由于500μl溶剂稀释75.6毫克4- ABAH制备4- ABAH（151.17克/摩尔）在二甲基亚砜（DMSO）的1M储备溶液。进一步稀释4 ABAH 1/10 HBSS。 </li></ol></li><li>用于制备H 2 O 2的工作液标签中的两个1.5毫升离心管中分别与"70毫米H 2 O 2"和"7毫米H 2 O 2"。 <ol><li>在标有"70毫米H 2 O 2"管，新鲜稀释10微升的使用990微升蒸馏水中，得到约88毫米的浓度的 H 2 O 2的30％储备溶液（8.8毫摩尔）。储存在冰上，4小时内使用。 注:H 2 O 2通常递送作为30％原液由供应商。当储存在4℃下，它是约3年的稳定。在蒸馏水中进行的H 2 O 2稀释液，以避免分解。 的 H 2 O 2稀释液也可在0.1M NaOH溶液中制备。 </li><li>对于确切H的决心2 O 2浓度由该解决方案到900μl，以1.5 ml管蒸馏水标记加入100微升从第一H准备再1至10稀释2 O 2原液（约88毫米） "7毫米H 2 O 2"。 </li><li>通过使用紫外-可见photospectrometer记录在近紫外范围（ 例如，200-300 nm）的光谱，并使用吸光度在240纳米（49; 240 = 34 M -1厘米-1），以确定实际的H 2 O 2浓度在第二H 2 O 2原液34。确保该参考比色杯仅含有蒸馏水。对于测量使用石英比色皿作为在UV范围内的光谱被记录。 </li><li>从该结果，计算两者的 H 2 O 2的储备溶液的确切浓度。通过加入蒸馏水适量调节第一原液的浓度，在"70毫的 H 2 O 2的"样品管恰好70毫米。储存在冰上将得到的工作溶液，并在一小时内使用。 </li></ol></li><li>加入5微升4- ABAH工作溶液（100毫米）到适当的样品进行1mM的终浓度。在APF加入之前培养箱孵育15分钟将样品在37℃。 </li><li>添加5微升的APF工作溶液（1毫摩尔），以每500微升样品，以获得10微米的最终染料浓度。轻轻混匀样品（ 例如，通过使用介质设置涡旋混合器），在37℃下孵育30分钟。 </li><li>添加5微升的 H 2 O 2的工作溶液（70毫摩尔），以每500微升样品为700微米的最终浓度。轻轻地混合样品，并在37℃孵育他们60分钟。并行执行适当控制措施。 注意:控制测量结果表明，700微米的 H 2 O 2的是没有毒性的细胞。任何显著细胞反应也不进行观察。 注:APF染色也可通过省略加入的 H 2 O 2，这取决于科学问题进行的。在不存在过氧化氢 ​​的仅基底氯化MPO和EPO活性而加入的 H 2 O 2的允许的最大次氯酸和次溴酸所生产的细胞的检测来确定。 ŧ他后者意味着一个显著更高的荧光信号。 </li><li>用于制粒的细胞，离心样品，在室温下400×g下10分钟和。彻底除去上清液而不破坏颗粒并添加250微升HBSS以悬浮细胞。 注:该样品现在已经准备好通过流式细胞仪分析25。 </li><li>存储在黑暗中的样品直至分析，以避免漂白。在480-490 nm激发后检测荧光素的发射约525纳米的37。 </li></ol>

The authors have nothing to disclose.

嗜中性粒细胞是人体血液中最丰富的白细胞的过氧化物酶阳性细胞的分离通常只着重于这些细胞，并包括由密度梯度离心38从其它白细胞嗜中性粒细胞的分离。然而，作为嗜中性粒细胞是鼠的血液样本中少得多的丰富39对后者更复杂的方法，必须使用40。而且这两种方法也导致除去从样品中的过氧化物酶阳性单核细胞的和，由于需要更大的血液体积（ 例如，400微升<su...

多形核白细胞（PMN，也称为粒细胞）和单核细胞代表在血液中1,2-先天免疫系统的重要细胞成分。它们向主防御病原体以及所获取的免疫系统的活化和全身性的炎症反应2-4的启动。然而尤其嗜中性粒细胞，最丰富的类型粒，和单核细胞也显著有助于急性炎性事件5的调节和终止。因此，这些细胞也可能在慢性炎性疾病如类风湿关节炎6,7-重要作用。事实上，哮喘，慢性炎性气道疾病，特点是嗜酸性粒细胞的削弱的细胞凋亡，在血液8的第二大粒型。然而粒细胞凋亡及快速拆卸由巨噬细胞是细胞终止在两个基本步骤炎症9-11。 在已命名的免疫细胞两个密切相关的酶，即髓过氧化物酶（MPO，中性粒细胞和单核细胞）和嗜酸性粒细胞过氧化物酶（EPO，嗜酸性粒细胞）可以发现12,13。这些血红素过氧化物酶古典相关，因为它们两个电子氧化（伪）卤化为相应的低（伪），其为它们的杀菌性能14-16已知亚卤酸体液免疫应答。在生理条件下的MPO主要形式次氯酸（次氯酸）和hypothiocyanite -而后者和次溴酸（次溴酸）由EPO 17-19形成（OSCN）。新的结果表明，这种（伪）卤化酶活性也可能有助于炎性反应的调节和免疫反应20,21的终止。事实上，次氯酸生产由MPO和衍生产品被证明抑制Ť基于细胞的适应性免疫应答22-24。 为了在慢性炎症性疾病，以获得从先天免疫系统更见解白细胞的免疫作用，并确定MPO和EPO的贡献这种生理功能，我们开发迅速丰富从小血样白细胞用于随后特定的方法测定这些细胞中的卤化过氧化物酶活性。为红细胞耗尽我们选择的标准方法，包括用蒸馏水，这导致了快速的白细胞富集在低材料成本的两后续低渗裂解步骤。对于卤化MPO随后的决心和EPO活动HOCl-和次溴酸专用染料荧光氨苯（APF）用于25-27。在对比的非特异性过氧化物酶染色方法28,29中的应用，这种方法使卤化过氧化物酶活性的选择性检测，这往往是在严重INFL障ammation 30,31。

<table><tbody><tr><td>&lt;em&gt;Materials/Equipment&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>15 ml centrifugation tubes</td><td>VWR/Corning</td><td>734-0451</td><td>-</td></tr><tr><td>1.5 ml sample tubes</td><td>VWR/Eppendorf</td><td>211-2130DE</td><td>-</td></tr><tr><td>Pipettes for volumes up to 5 ml</td><td>Eppendorf</td><td>&lt;em&gt;e.g.&lt;/em&gt;, 3120000070</td><td>We are using Eppendorf Resarch plus pipettes with adjustable volumes in the range 1-10 &amp;micro;l, 10-100 &amp;micro;l, 100-1,000 &amp;micro;l an 500-5,000 &amp;micro;l</td></tr><tr><td>laminar flow bench</td><td>Thermo Electron Corperation</td><td>HeraSafe</td><td>-</td></tr><tr><td>Vortex mixer</td><td>Bender &amp;amp; Hobein AG</td><td>Vortex Genie 2</td><td>-</td></tr><tr><td>Tabletop centrifuge</td><td>Kendro Laboratory Products</td><td>Laborfuge 400R</td><td>The centrifuge should be able to be used at 450 x g</td></tr><tr><td>Small centrifuge</td><td>Eppendorf</td><td>5415D</td><td>The centrifuge should be able to be used at 400 x g</td></tr><tr><td>Incubator</td><td>Heraeus</td><td>cytoperm 2</td><td>Settings: 37 &amp;deg;C, 95% humidity, 5% CO&lt;sub&gt;2&lt;/sub&gt; content</td></tr><tr><td>UV-Vis spectrophotometer</td><td>Varian</td><td>Cary 50 bio</td><td>A spectrum between 200 and 300 nm has to be recorded. Thus quartz cuvettes have to be applied</td></tr><tr><td>Flow cytometer</td><td>Becton, Dickinson</td><td>BD Facs Calibur</td><td>Any flow cytometer can be used which is equiped with a laser suitable for the excitation of fluorescein (&lt;em&gt;e.g.&lt;/em&gt;, 488 nm argon laser)</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;em&gt;Chemicals&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>amresco</td><td>K812</td><td>sterile solution, ready to use</td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Sigma-Aldrich</td><td>P4417</td><td>tablets for solving in 200 ml millipore water</td></tr><tr><td>Hanks balanced salt solution (HBSS) with Ca&lt;sup&gt;2+&lt;/sup&gt;</td><td>Sigma-Aldrich</td><td>H1387</td><td>970 mg/100 ml, carefully check and adjust the pH value to 7.4</td></tr><tr><td>Hydrochloric acid</td><td>Merck Millipore</td><td>1.09057.1000</td><td>1 M solution</td></tr><tr><td>Sodium hydroxide</td><td>Riedel-deHa&amp;euml;n</td><td>30620</td><td>Solid pellets. For a 1 M solution solve 4 g/100 ml Millipore water</td></tr><tr><td>Aminophenyl fluorescein</td><td>Cayman</td><td>10157</td><td>5 mg/ml solution (11.81 mM) in methyl acetate, aliquots (&lt;em&gt;e.g.&lt;/em&gt; 100 &amp;micro;l) should be prepared and stored at -20 &amp;deg;C</td></tr><tr><td>Hydrogen peroxide</td><td>Sigma-Aldrich</td><td>H1009</td><td>This 30% stock solution corresponds to a concentration of about 8.8 M. Further dilutions have to be freshly prepared in distilled water immediately prior to use and quantified by absorbance measurements</td></tr><tr><td>4-aminobenzoic acid hydrazide (4-ABAH)</td><td>Sigma-Aldrich</td><td>A41909</td><td>A first stock solution of 1 M should be prepared in DMSO a second one of 100 mM by 1:10 dilution in HBSS</td></tr><tr><td>DMSO</td><td>VWR chemicals</td><td>23500.26</td><td>-</td></tr></tbody></table>

fast and specific assessment of the halogenating peroxidase activity in leukocyte-enriched blood samples

本文从小型全血样本中的白细胞快速，标准化富集的协议描述。这个过程是基于红细胞的低渗裂解和可以应用到人类的样本以及非人类来源的血液。的约50至100微升小初始样品体积使得这种方法适用于反复采血从小型实验室动物。此外，白细胞富集分钟之内，并与有关化学品和仪器仪表材料低的努力所取得，使得这种方法适用于多个实验室环境。 白细胞的标准化纯化是用高选择性染色法来评价血红素过氧化物酶的卤化过氧化物酶，髓过氧化物酶（MPO）和嗜酸性粒细胞过氧化物酶（EPO）， 即，次氯酸和次溴酸（次氯酸和次溴酸）的形成相结合。虽然MPO是NEUT强烈表达rophils，在人血液中，以及在单核细胞中最丰富的免疫细胞类型，相关的酶的EPO是专门在嗜酸性粒细胞中表达。这些酶的卤化活性是通过使用几乎HOCl-和次溴酸特异性染料氨基苯基荧光素（APF）和主过氧化物过氧化衬底氢处理。在随后的流式细胞分析所有过氧化物酶阳性细胞（嗜中性粒细胞，单核细胞，嗜酸性粒细胞）的区分和它们的卤化过氧化物酶的活性可以被量化。因为APF染色可以与细胞表面标记物的应用相结合，这个协议可以扩展到专门处理白细胞亚级分。该方法适用于同时检测在人类和啮齿动物白细胞次氯酸和次溴酸的生产。 鉴于这些酶产品在慢性炎症性疾病的广泛和多样讨论免疫的作用，这个协议可能有助于更好地理解免疫白细胞衍生的血红素的过氧化物酶的相关性。

如先前报道上述方法原来是既适用于人类和非人类材料32。此外如图用于与哮喘症状的小鼠将APF染色可检测的全身促炎状态的差异的合适的工具。因此，在我们使用该协议随后的研究中反复雌性黑刺鼠大鼠降植烷诱导关节炎（PIA）评估MPO（和EPO）的卤化活动。白细胞富集和该实验期间进行染色的代表性实例示于图1。在麻醉下从动物的眼球后静脉...

Watch this Scientific Journal Video about Fast and Specific Assessment of the Halogenating Peroxidase Activity in Leukocyte-enriched Blood Samples at JoVE.com

Fast and Specific Assessment of the Halogenating Peroxidase Activity in Leukocyte-enriched Blood Samples

This protocol describes the quick enrichment of leukocytes from small blood samples for a subsequent specific determination of the halogenating peroxidase activity within the cells. The method can be applied to human and non-human material and may contribute to the evaluation of new inflammatory markers.

在富含白细胞的血液样本卤化过氧化物酶活性的快速，具体的评估

In this paper a protocol for the quick and standardized enrichment of leukocytes from small whole blood samples is described. This procedure is based on ...

fast-specific-assessment-halogenating-peroxidase-activity-leukocyte

Research

JoVE Journal

Immunology and Infection

10.3K 次观看.  University of Leipzig. This protocol describes the quick enrichment of leukocytes from small blood samples for a subsequent specific determination of the halogenating peroxidase activity within the cells. The method can be applied to human and non-human material and may contribute to the evaluation of new inflammatory markers.

视频: Fast and Specific Assessment of the Halogenating Peroxidase Activity in Leukocyte-enriched Blood Samples

Imaging Approaches to Assessments of Toxicological Oxidative Stress Using Genetically-encoded Fluorogenic Sensors

While oxidative stress is a commonly cited toxicological mechanism, conventional methods to study it suffer from a number of shortcomings, including destruction of the sample, introduction of potential artifacts, and a lack of specificity for the reactive species involved. Thus, there is a current need in the field of toxicology for non-destructive, sensitive, and specific methods that can be used to observe and quantify intracellular redox perturbations, more commonly referred to as oxidative stress. Here, we present a method for the use of two genetically-encoded fluorogenic sensors, roGFP2 and HyPer, to be used in live-cell imaging studies to observe xenobiotic-induced oxidative responses. roGFP2 equilibrates with the glutathione redox potential (EGSH), while HyPer directly detects hydrogen peroxide (H2O2). Both sensors can be expressed into various cell types via transfection or transduction, and can be targeted to specific cellular compartments. Most importantly, live-cell microscopy using these sensors offers high spatial and temporal resolution that is not possible using conventional methods. Changes in the fluorescence intensity monitored at 510 nm serves as the readout for both genetically-encoded fluorogenic sensors when sequentially excited by 404 nm and 488 nm light. This property makes both sensors ratiometric, eliminating common microscopy artifacts and correcting for differences in sensor expression between cells. This methodology can be applied across a variety of fluorometric platforms capable of exciting and collecting emissions at the prescribed wavelengths, making it suitable for use with confocal imaging systems, conventional wide-field microscopy, and plate readers. Both genetically-encoded fluorogenic sensors have been used in a variety of cell types and toxicological studies to monitor cellular EGSH and H2O2 generation in real-time. Outlined here is a standardized method that is widely adaptable across cell types and fluorometric platforms for the application of roGFP2 and HyPer in live-cell toxicological assessments of oxidative stress.

This manuscript describes the use of genetically-encoded fluorogenic reporters in an application of live-cell imaging for the examination of xenobiotic-induced oxidative stress. This experimental approach offers unparalleled spatiotemporal resolution, sensitivity, and specificity while avoiding many of the shortcomings of conventional methods used for the detection of toxicological oxidative stress.

用基因编码荧光传感器评估毒理学氧化应激的成像方法

While oxidative stress is a commonly cited toxicological mechanism, conventional methods to study it suffer from a number of shortcomings, including ...

科研

癌症研究

Detection of Total Reactive Oxygen Species in Adherent Cells by 2',7'-Dichlorodihydrofluorescein Diacetate Staining

Oxidative stress is an important event under both physiological and pathological conditions. In this study, we demonstrate how to quantify oxidative stress by measuring total reactive oxygen species (ROS) using 2&#39;,7&#39;-dichlorodihydrofluorescein diacetate (DCFH-DA) staining in colorectal cancer cell lines as an example. This protocol describes detailed steps including preparation of DCFH-DA solution, incubation of cells with DCFH-DA solution, and measurement of normalized intensity. DCFH-DA staining is a simple and cost-effective way to detect ROS in cells. It can be used to measure ROS generation after chemical treatment or genetic modifications. Therefore, it is useful for determining cellular oxidative stress upon environment stress, providing clues to mechanistic studies.

Here, we present a protocol to detect total cellular reactive oxygen species (ROS) using 2&#39;,7&#39;-dichlorodihydrofluorescein diacetate (DCFH-DA). This method can visualize cellular ROS localization in adherent cells with a fluorescence microscope and quantify ROS intensity with a fluorescence plate reader. This protocol is simple, efficient and cost-effective.

通过2'，7'-二氯二氢氟辛二乙酸染色检测粘附细胞中的总活性氧物种

Oxidative stress is an important event under both physiological and pathological conditions. In this study, we demonstrate how to quantify oxidative ...

生物化学

Bioenergetics and the Oxidative Burst: Protocols for the Isolation and Evaluation of Human Leukocytes and Platelets

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and neurodegenerative diseases but assessing changes in bioenergetic function in patients is challenging.&#160;Although diseases such as diabetes or atherosclerosis present clinically with specific organ impairment, the systemic components of the pathology, such as hyperglycemia or inflammation, can alter bioenergetic function in circulating leukocytes or platelets. This concept has been recognized for some time but its widespread application has been constrained by the large number of primary cells needed for bioenergetic analysis.&#160;This technical limitation has been overcome by combining the specificity of the magnetic bead isolation techniques, cell adhesion techniques, which allow cells to be attached without activation to microplates, and the sensitivity of new technologies designed for high throughput microplate respirometry.&#160;An example of this equipment is the extracellular flux analyzer. Such instrumentation typically uses oxygen and pH sensitive probes to measure rates of change in these parameters in adherent cells, which can then be related to metabolism. Here we detail the methods for the isolation and plating of monocytes, lymphocytes, neutrophils and platelets, without activation, from human blood and the analysis of mitochondrial bioenergetic function in these cells. In addition, we demonstrate how the oxidative burst in monocytes and neutrophils can also be measured in the same samples. Since these methods use only 8-20 ml human blood they have potential for monitoring reactive oxygen species generation and bioenergetics in a clinical setting.

Blood leukocytes and platelets can be used as a marker of overall bioenergetic health of an individual and so have the potential to monitor pathological processes and the impact of treatments. Here&#160;we describe a method to isolate and measure mitochondrial function and the oxidative burst in these cells.

生物能量和氧化突发：协议用于人类白细胞和血小板的隔离与评价

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and ...

免疫与感染

Hellgrammite as a Non-Conventional Biomonitor for Surface Water and Environmental Health Assessment

The larvae of dobsonflies from the genus Corydalus, commonly known as Hellgrammites, are characterized by their notable size, extensive range of occurrence, and extended period of immaturity, which can last up to one year. Hellgrammites are clearly known to exhibit sensitivity to pollution and habitat structure impacts. Given these unique features, using Corydalus texanus larvae is highly suitable as reliable biomonitoring agents to assess the ecological integrity of aquatic ecosystems. This protocol aims to provide the necessary tools for C. texanus assessment and demonstrate their efficacy through a case study. Research findings have practical implications, indicating that C. texanus larvae exhibit early-warning responses to mining pollution, bioaccumulating high amounts of heavy metals such as Zn, Fe, and Al. The presence or absence of C. texanus populations may serve as a helpful indicator for identifying potential issues related to ecosystem health. The unconventional approach has shown early warnings of pollution in mining-impacted sites, highlighting the need for timely action to protect the environment. Given their unique traits, the use of C. texanus larvae is highly suggested as a reliable non-conventional bioindicator.

This protocol describes the use of biomarkers for the early detection of deleterious impacts in aquatic ecosystems. The biomarkers are closely related to sentinel traits, and their changes aid in detecting early-warning damages.

Hellgrammite 作为地表水和环境健康评估的非常规生物监测器

The larvae of dobsonflies from the genus Corydalus, commonly known as Hellgrammites, are characterized by their notable size, extensive range of ...

环境科学

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 ...

Detecting, Visualizing and Quantitating the Generation of Reactive Oxygen Species in an Amoeba Model System

Reactive oxygen species (ROS) comprise a range of reactive and short-lived, oxygen-containing molecules, which are dynamically interconverted or eliminated either catalytically or spontaneously. Due to the short life spans of most ROS and the diversity of their sources and subcellular localizations, a complete picture can be obtained only by careful measurements using a combination of protocols. Here, we present a set of three different protocols using OxyBurst Green (OBG)-coated beads, or dihydroethidium (DHE) and Amplex UltraRed (AUR), to monitor qualitatively and quantitatively various ROS in professional phagocytes such as Dictyostelium. We optimised the beads coating procedures and used OBG-coated beads and live microscopy to dynamically visualize intraphagosomal ROS generation at the single cell level. We identified lipopolysaccharide (LPS) from E. coli as a potent stimulator for ROS generation in Dictyostelium. In addition, we developed real time, medium-throughput assays using DHE and AUR to quantitatively measure intracellular superoxide and extracellular H2O2 production, respectively.

We adapted a set of protocols for the measurement of reactive oxygen species (ROS) that can be applied in various amoeba and mammalian cellular models for qualitative and quantitative studies.

检测，可视化和定量活性氧的生成在变形虫模型系统

Reactive oxygen species (ROS) comprise a range of reactive and short-lived, oxygen-containing molecules, which are dynamically interconverted or ...

生物学

<meta charset="utf8"></head><body>使用荧光成像和 EPR 检测血小板中的超氧阴离子

Alternative Methods for the Detection of Superoxide Anion Generation in Platelets

Reactive oxygen species (ROS) are highly unstable oxygen-containing molecules. Their chemical instability makes them extremely reactive and gives them the ability to react with important biological molecules such as proteins, nucleic acids, and lipids. Superoxide anions are important ROS generated by the reduction of molecular oxygen reduction (i.e., acquisition of one electron). Despite their initial implication exclusively in aging, degenerative, and pathogenic processes, their participation in important physiological responses has recently become apparent. In the vascular system, superoxide anions have been shown to modulate the differentiation and function of vascular smooth muscle cells, the proliferation and migration of vascular endothelial cells in angiogenesis, the immune response, and the activation of platelets in hemostasis. The role of superoxide anions is particularly important in the dysregulation of platelets and the cardiovascular complications associated with a plethora of conditions, including cancer, infection, inflammation, diabetes, and obesity. It has, therefore, become extremely relevant in cardiovascular research to be able to effectively measure the generation of superoxide anions by&#160;human platelets,&#160;understand the redox-dependent mechanisms regulating the balance between hemostasis and thrombosis and, eventually,&#160;identify novel pharmacological tools for the modulation of platelet responses leading to thrombosis and cardiovascular complications. This study presents three experimental protocols successfully adopted for the detection of superoxide anions in platelets and the study of the redox-dependent mechanisms regulating hemostasis and thrombosis: 1) dihydroethidium (DHE)-based superoxide anion detection by flow cytometry; 2) DHE-based superoxide anion visualization and analysis by single platelet imaging; and 3) spin probe-based quantification of superoxide anion output in platelets by electron paramagnetic resonance (EPR).

<meta charset="utf8"></head><body>作者聚焦：ROS 检测的创新技术及其对血小板研究的影响

The generation of superoxide anion is essential for the stimulation of platelets and, if dysregulated, critical for thrombotic diseases. Here, we present three protocols for the selective detection of superoxide anions and the study of redox-dependent platelet regulation.

检测血小板中超氧阴离子生成的替代方法 

Reactive oxygen species (ROS) are highly unstable oxygen-containing molecules. Their chemical instability makes them extremely reactive and gives them the ...

Evaluation of a Point-of-Care Testing Analyzer for Measuring Peripheral Blood Leukocytes

White blood cell (WBC) is an important indicator of inflammation in the body, and it can help distinguish between bacterial and viral infections. At present, most primary medical institutions in China have a poor percentage of adoption of blood-testing technology, and a hematology detection system with a high price to performance ratio and easy operation is urgently needed in primary healthcare centers. This paper introduces the principle and operation procedures of a point-of-care testing (POCT) card-based leukocyte analyzer (evaluated system), which was used to detect WBC indexes such as neutrophils, lymphocytes, and intermediate group cells (including eosinophils, basophils, and monocytes) in whole blood. The results from the evaluated system were compared to those from two commercial automatic hematology analyzers (reference system). The correlation and consistency between the evaluated system and the commercial reference systems were analyzed. The results showed that WBC count and number of granulocytes detected by the evaluated and reference systems showed a strong positive correlation (rs = 0.972 and 0.973, respectively), while the number of lymphocytes showed a relatively low correlation (rs = 0.851). A Bland-Altman plot showed that the major difference between the values detected by the evaluated system and the reference systems is within 95% limits of agreement (LoA), indicating that the two systems are in good agreement. In conclusion, the evaluated system has an excellent correlation, robust consistency, and a reliable comparison with the results of the widely used automatic hematology analyzers. It is ideal for WBC detection in primary medical institutions where a full-automatic five-category hematology analyzer is unavailable, especially during the COVID-19 normalized prevention and control period.

A protocol to measure peripheral blood leukocytes using a POCT card-based leukocyte analyzer is presented here. Same blood samples were tested by two automated hematology analyzers to evaluate the consistency and accuracy of the results. The results showed that the evaluated analyzer had a good correlation with the reference system.

用于测量外周血白细胞的床旁测试分析仪的评估

White blood cell (WBC) is an important indicator of inflammation in the body, and it can help distinguish between bacterial and viral infections. At ...

在富含白细胞的血液样本卤化过氧化物酶活性的快速，具体的评估

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此视频中的章节

用基因编码荧光传感器评估毒理学氧化应激的成像方法

通过2'，7'-二氯二氢氟辛二乙酸染色检测粘附细胞中的总活性氧物种

生物能量和氧化突发：协议用于人类白细胞和血小板的隔离与评价

Hellgrammite 作为地表水和环境健康评估的非常规生物监测器

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

检测，可视化和定量活性氧的生成在变形虫模型系统

检测血小板中超氧阴离子生成的替代方法

检测血小板中超氧阴离子生成的替代方法

检测血小板中超氧阴离子生成的替代方法

用于测量外周血白细胞的床旁测试分析仪的评估