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 border="0" cellpadding="0" cellspacing="0" width="738">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">materials/equipment</td>
			<td style="width:203px;"></td>
			<td style="width:120px;"></td>
			<td style="width:203px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">15 ml centrifugation tubes</td>
			<td style="width:203px;">VWR/Corning</td>
			<td style="width:120px;">734-0451</td>
			<td style="width:203px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">1.5 ml sample tubes</td>
			<td style="width:203px;">VWR/Eppendorf</td>
			<td style="width:120px;">211-2130DE</td>
			<td style="width:203px;">-</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:213px;">Pipettes for volumes up to 5 ml</td>
			<td style="width:203px;">Eppendorf</td>
			<td style="width:120px;">e.g. 3120000070</td>
			<td style="width:203px;">We are using Eppendorf Resarch plus pipettes with adjustable volumes in the range 1-10 &#181;l, 10-100&#181;l, 100-1000 &#181;l an 500-5000&#181;l</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">laminar flow bench</td>
			<td style="width:203px;">Thermo Electron Corperation</td>
			<td style="width:120px;">HeraSafe</td>
			<td style="width:203px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Vortex mixer</td>
			<td style="width:203px;">Bender &#38; Hobein AG</td>
			<td style="width:120px;">Vortex Genie 2</td>
			<td style="width:203px;">-</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Tabletop centrifuge</td>
			<td style="width:203px;">Kendro Laboratory Products</td>
			<td style="width:120px;">Laborfuge 400R</td>
			<td style="width:203px;">The centrifuge should be able to be used at 450x g</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Small centrifuge</td>
			<td style="width:203px;">eppendorf</td>
			<td style="width:120px;">5415D</td>
			<td style="width:203px;">The centrifuge should be able to be used at 400x g</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Incubator</td>
			<td style="width:203px;">Heraeus</td>
			<td style="width:120px;">cytoperm 2</td>
			<td style="width:203px;">Settings: 37 &#176;C, 95% humidity, 5 % CO2 content</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">UV-Vis spectrophotometer</td>
			<td style="width:203px;">Varian</td>
			<td style="width:120px;">Cary 50 bio</td>
			<td style="width:203px;">A spectrum between 200 and 300 nm has to be recorded. Thus quartz cuvettes have to be applied</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;">Flow cytometer</td>
			<td style="width:203px;">Becton, Dickinson</td>
			<td style="width:120px;">BD Facs Calibur</td>
			<td style="width:203px;">Any flow cytometer can be used which is equiped with a laser suitable for the excitation of fluorescein (e.g 488 nm argon laser)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td style="width:203px;">Company</td>
			<td style="width:120px;">Catalog Number</td>
			<td style="width:203px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Chemicals</td>
			<td style="width:203px;"></td>
			<td style="width:120px;"></td>
			<td style="width:203px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:213px;">Phosphate buffered saline (PBS)</td>
			<td style="width:203px;">amresco</td>
			<td style="width:120px;">K812</td>
			<td style="width:203px;">sterile solution, ready to use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:213px;"></td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:120px;">P4417</td>
			<td style="width:203px;">tablets for solving in 200 ml millipore water</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:213px;">Hanks balanced salt solution (HBSS) with Ca(2+)</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:120px;">H1387</td>
			<td style="width:203px;">970 mg/100 ml, carefully check and adjust the pH value to 7.4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Hydrochloric acid</td>
			<td style="width:203px;">Merck Millipore</td>
			<td style="width:120px;">1.09057.1000</td>
			<td style="width:203px;">1M solution</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:213px;">Sodium hydroxide</td>
			<td style="width:203px;">Riedel-deHa&#235;n</td>
			<td style="width:120px;">30620</td>
			<td style="width:203px;">Solid pellets. For a 1M solution solve 4 g/100 ml Millipore water</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:213px;">Aminophenyl fluorescein</td>
			<td style="width:203px;">Cayman</td>
			<td style="width:120px;">10157</td>
			<td style="width:203px;">5 mg/ml solution (11.81 mM) in methyl acetate, aliquotes of e.g. 100 &#181;l should be prepared and stored at -20 &#176;C</td>
		</tr>
		<tr height="168">
			<td height="168" style="height:168px;width:213px;">Hydrogen peroxide</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:120px;">H1009</td>
			<td style="width:203px;">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 height="84">
			<td height="84" style="height:84px;width:213px;">4-aminobenzoic acid hydrazide (4-ABAH)</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:120px;">A41909</td>
			<td style="width:203px;">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 height="21">
			<td height="21" style="height:21px;width:213px;">DMSO</td>
			<td style="width:203px;">VWR chemicals</td>
			<td style="width:120px;">23500.26</td>
			<td style="width:203px;">-</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

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JoVE Journal

Immunology and Infection

10.2K 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

Depletion of Specific Cell Populations by Complement Depletion

The purification of immune cell populations is often required in order to study their unique functions. In particular, molecular approaches such as real-time PCR and microarray analysis require the isolation of cell populations with high purity. Commonly used purification strategies include fluorescent activated cell sorting (FACS), magnetic bead separation and complement depletion. Of the three strategies, complement depletion offers the advantages of being fast, inexpensive, gentle on the cells and a high cell yield. The complement system is composed of a large number of plasma proteins that when activated initiate a proteolytic cascade culminating in the formation of a membrane-attack complex that forms a pore on a cell surface resulting in cell death1. The classical pathway is activated by IgM and IgG antibodies and was first described as a mechanism for killing bacteria. With the generation of monoclonal antibodies (mAb), the complement cascade can be used to lyse any cell population in an antigen-specific manner. Depletion of cells by the complement cascade is achieved by the addition of complement fixing antigen-specific antibodies and rabbit complement to the starting cell population. The cells are incubated for one hour at 37&deg;C and the lysed cells are subsequently removed by two rounds of washing. MAb with a high efficiency for complement fixation typically deplete 95-100% of the targeted cell population. Depending on the purification strategy for the targeted cell population, complement depletion can be used for cell purification or for the enrichment of cell populations that then can be further purified by a subsequent method.

To effectively study the function of immune cell populations their purification is often required. Complement depletion is a fast and inexpensive technique for the isolation of immune cell populations with high purity.

补消耗的特定细胞群的枯竭

The purification of immune cell populations is often required in order to study their unique functions.  In particular, molecular approaches such as ...

科研

免疫与感染

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known phenotype.  Other bulk methods of purification include panning, complement depletion and magnetic bead separation.  However, FACS has several advantages over other available methods.  FACS is the preferred method when very high purity of the desired population is required, when the target cell population expresses a very low level of the identifying marker or when cell populations require separation based on differential marker density.  In addition, FACS is the only available purification technique to isolate cells based on internal staining or intracellular protein expression, such as a genetically modified fluorescent protein marker.  FACS allows the purification of individual cells based on size, granularity and fluorescence.  In order to purify cells of interest, they are first stained with fluorescently-tagged monoclonal antibodies (mAb), which recognize specific surface markers on the desired cell population (1).  Negative selection of unstained cells is also possible.  FACS purification requires a flow cytometer with sorting capacity and the appropriate software.  For FACS, cells in suspension are passed as a stream in droplets with each containing a single cell in front of a laser.  The fluorescence detection system detects cells of interest based on predetermined fluorescent parameters of the cells.  The instrument applies a charge to the droplet containing a cell of interest and an electrostatic deflection system facilitates collection of the charged droplets into appropriate collection tubes (2).  The success of staining and thereby sorting depends largely on the selection of the identifying markers and the choice of mAb.  Sorting parameters can be adjusted depending on the requirement of purity and yield.  Although FACS requires specialized equipment and personnel training, it is the method of choice for isolation of highly purified cell populations.

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting FACS

For many scientific studies requiring a biological and chemical analysis of cell populations the cells must be in a high state of purity. Fluorescence activated cell sorting (FACS) is a superior method in which to obtain pure cell populations.

特定细胞群的净化荧光激活细胞分选（FACS）

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

在细菌粘附的研究剪应力

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Determining the Phagocytic Activity of Clinical Antibody Samples

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as pathology of disease. While the properties of the variable domains of antibodies have long been considered critical to in vivo function, the ability of antibodies to recruit innate immune cells via their Fc domains has become increasingly appreciated as a major factor in their efficacy, both in the setting of recombinant monoclonal antibody therapy, as well as in the course of natural infection or vaccination1-3.
Importantly, despite its nomenclature as a constant domain, the antibody Fc domain does not have constant function, and is strongly modulated by IgG subclass (IgG1-4) and glycosylation at Asparagine 2974-6. Thus, this method to study functional differences of antigen-specific antibodies in clinical samples will facilitate correlation of the phagocytic potential of antibodies to disease state, susceptibility to infection, progression, or clinical outcome.
Furthermore, this effector function is particularly important in light of the documented ability of antibodies to enhance infection by providing pathogens access into host cells via Fc receptor-driven phagocytosis7. Additionally, there is some evidence that phagocytic uptake of immune complexes can impact the Th1/Th2 polarization of the immune response8.
Here, we describe an assay designed to detect differences in antibody-induced phagocytosis, which may be caused by differential IgG subclass, glycan structure at Asn297, as well as the ability to form immune complexes of antigen-specific antibodies in a high-throughput fashion. To this end, 1 &mu;m fluorescent beads are coated with antigen, then incubated with clinical antibody samples, generating fluorescent antigen specific immune complexes. These antibody-opsonized beads are then incubated with a monocytic cell line expressing multiple Fc&gamma;Rs, including both inhibitory and activating. Assay output can include phagocytic activity, cytokine secretion, and patterns of Fc&gamma;Rs usage, and are determined in a standardized manner, making this a highly useful system for parsing differences in this antibody-dependent effector function in both infection and vaccine-mediated protection9.

We present a high-throughput flow cytometric assay to determine the phagocytic activity of antigen-specific antibodies from clinical samples, utilizing fluorescent antigen-coated beads and a monocytic cell line expressing multiple Fc receptors&#x2014;providing receptor usage and phagocytic activity determinations in a standardized and reproducible fashion for any antigen of interest.

确定临床抗体样品的吞噬活性

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as ...

Quantitative Imaging of Lineage-specific Toll-like Receptor-mediated Signaling in Monocytes and Dendritic Cells from Small Samples of Human Blood

Individual variations in immune status determine responses to infection and contribute to disease severity and outcome. Aging is associated with an increased susceptibility to viral and bacterial infections and decreased responsiveness to vaccines with a well-documented decline in humoral as well as cell-mediated immune responses1,2. We have recently assessed the effects of aging on Toll-like receptors (TLRs), key components of the innate immune system that detect microbial infection and trigger antimicrobial host defense responses3. In a large cohort of healthy human donors, we showed that peripheral blood monocytes from the elderly have decreased expression and function of certain TLRs4 and similar reduced TLR levels and signaling responses in dendritic cells (DCs), antigen-presenting cells that are pivotal in the linkage between innate and adaptive immunity5. We have shown dysregulation of TLR3 in macrophages and lower production of IFN by DCs from elderly donors in response to infection with West Nile virus6,7. 
Paramount to our understanding of immunosenescence and to therapeutic intervention is a detailed understanding of specific cell types responding and the mechanism(s) of signal transduction. Traditional studies of immune responses through imaging of primary cells and surveying cell markers by FACS or immunoblot have advanced our understanding significantly, however, these studies are generally limited technically by the small sample volume available from patients and the inability to conduct complex laboratory techniques on multiple human samples. ImageStream combines quantitative flow cytometry with simultaneous high-resolution digital imaging and thus facilitates investigation in multiple cell populations contemporaneously for an efficient capture of patient susceptibility. Here we demonstrate the use of ImageStream in DCs to assess TLR7/8 activation-mediated increases in phosphorylation and nuclear translocation of a key transcription factor, NF-&kappa;B, which initiates transcription of numerous genes that are critical for immune responses8. Using this technology, we have also recently demonstrated a previously unrecognized alteration of TLR5 signaling and the NF-&kappa;B pathway in monocytes from older donors that may contribute to altered immune responsiveness in aging9.

We describe use of ImageStream technology (<a href="http://www.amnis.com/" target="_blank">www.amnis.com</a>), which combines quantitative flow cytometry with simultaneous high-resolution digital imaging, to quantify cellular mechanisms of primary immune cells from well-defined patient cohorts. Our studies provide a blueprint for translational investigations to quantify lineage specific cellular responses in small samples from subject cohorts.

定量成像小样本人体血液中单核细胞和树突状细胞系特定的Toll样受体介导的信号

Individual variations in immune status determine responses to infection and contribute to disease severity and outcome.  Aging is associated with an ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

从外周血人类同种异体抗原特异性T细胞的生成

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 &#181;m in diameter in inhalable particulate matter (PM10, particle size &#8804;10 &#181;m in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions.
So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles &#62;0.5 &#181;m.

Here, we present a protocol to quantify allergen-loaded particles by flow cytometry. Ambient particulate matter particles may act as carriers of adsorbed allergens. We show here that flow cytometry, a method widely used to characterize suspended solids &#62;0.5 &#181;m in diameter, can be used to measure these allergen-loaded particles.

粒子结合Bet v 1的变应原PM10的流式细胞分析

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

人全血中NK细胞杀伤活性的流式细胞仪分析

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

葡聚糖增强小鼠和人原发性 NK 细胞的慢转导效率

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

Trichinellosis is a debilitating disease in humans and is caused by the consumption of raw or undercooked meat of animals infected with the nematode larvae of the genus Trichinella. The most important sources of human infections worldwide are game meat and pork or pork products. In many countries, the prevention of human trichinellosis is based on the identification of infected animals by means of the artificial digestion of muscle samples from susceptible animal carcasses.
There are several methods based on the digestion of meat but the magnetic stirrer method is considered the gold standard. This method allows the detection of Trichinella larvae by microscopy after the enzymatic digestion of muscle samples and subsequent filtration and sedimentation steps. Although this method does not require special and expensive equipment, internal controls cannot be used. Therefore, stringent quality management should be applied throughout the test. The aim of the present work is to provide detailed handling instructions and critical control points of the method to analysts, based on the experience of the European Union Reference Laboratory for Parasites and the National Reference Laboratory of Germany for Trichinella.

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

The prevention of human trichinellosis in many countries worldwide is based on the laboratory examination of muscle samples from susceptible animals by methods of digestion, of which the magnetic stirrer method is considered the gold standard.

对于检测磁力搅拌器方法<em&gt;旋毛虫</em&gt;幼虫肌肉样品

Trichinellosis is a debilitating disease in humans and is caused by the consumption of raw or undercooked meat of animals infected with the nematode ...