This project was supported by Agriculture and Food Research Initiative Competitive Grant no. 2100-68003-30395 from the USDA National Institute of Food and Agriculture.

注:所有采血程序是按照由阿巴拉契亚州立大学（ASU）机构审查委员会（IRB）规定​​的准则进行。 1.全采血车<ol><li>有一个认证抽血根据世界卫生组织的指引抽血。 </li><li>抽血到含有二钾乙二胺四乙酸14毫升采血管（K 2 EDTA）。根据制造商的说明倒置血液收集管。保持采血管在室温下在台式摇杆，直到分离。 </li></ol> 2. DIO-标记的靶细胞的制备<ol><li>生长K562细胞中完整的Iscove氏改良的Dulbecco氏培养基（IMDM）补充有10％胎牛血清（FBS）和1％青霉素链霉素为之前测定，以保证细胞的健康几个星期。调整concentrat细胞的离子至3×10 5个细胞/ mL每日通过的小区的完成计数和细胞随后通过。执行完整的媒体变化每2至3天。 </li><li> 在测定当天，执行使用1细胞计数:1血细胞计数。 <ol><li>从K562烧瓶除去10μL的并放入1.5毫升管中。 </li><li>加台盼蓝染料的10微升到同为1.5毫升管为1:1的稀释因子:1。 </li><li>轻轻一抖管混合K562细胞台盼蓝染料。 </li><li>允许的K562细胞的台盼蓝染料孵育在室温下1分钟。 </li><li>从管中取出染K562细胞的15微升。 </li><li>吸移管到血球细胞计数。 </li><li>算在四角方块细胞以及总共五个方块的中间正方形。 </li><li>通过使用方程式计算细胞计数: 总细胞/ mL =总细胞数×（稀释倍数/平方数）×10000个/毫升</li></ol></l我&gt; <li>重悬的K562靶细胞以无血清IMDM培养基1×10 6个细胞/ ml的终密度。 </li><li>对未染色的K562靶细胞，以1×10 6个细胞/ mL的密度加10毫升的K562靶细胞于一个15毫升管，共10×10 6个 K562细胞。放入37℃培养箱中，用5％ 的 CO 2，直到准备使用。 </li><li> 对于DIO染的K562靶细胞，以1×10 6个细胞/ mL的密度加10毫升的K562靶向细胞于一个15毫升管，共10×10 6个 K562细胞。 <ol><li>添加细胞悬浮液的每毫升1 DIOμL的细胞标记溶液中指定为DIO染色，轻轻涡旋15ml试管。例如，以1×10 6个细胞/ mL的量添加的DIO细胞标记溶液10微升至10×10 6个 K562细胞/ ml。 </li><li>在一个15毫升管20分钟孵育的K562-DIO溶液，在37℃，5％的CO 2。 </li></ol></li><li>跟随ING孵育，添加3毫升室温磷酸盐缓冲盐水（PBS），以含有15毫升管的K562-DIO溶液。 </li><li>在室温下以135 xg离心离心10分钟。 </li><li>小心取出上清液，而不会干扰与1000微升音量可调移液器细胞沉淀。 </li><li>加入10 mL新鲜IMDM至15毫升管细胞含丸。 </li><li>轻轻涡管向悬浮细胞。 </li><li>重复步骤2.7至2.10两次。 </li><li>商店的细胞在37℃培养箱，用5％ 的 CO 2，直到准备使用。 注:细胞可以存储在培养箱长达24小时，但它是优选使用它们在同一天。 </li></ol> 3.控制的制备<ol><li> 传输以下为单独的和适当的标记好的1.5毫升管: <ol><li>补充新鲜IMDM 500微升含有悬浮DIO标记的K562细胞进入"双重利好"标记1.5毫升管。 </li><li>加入新鲜的IMDM 500μL含有悬浮DIO标记的K562细胞进入"仅DIO"标记1.5毫升管。 </li><li>添加含悬浮的未标记的K562细胞到新鲜的IMDM的500μL"碘化丙锭（PI）的唯一的"标记的1.5mL管中。 </li></ol></li><li>放置双阳性和PI仅管在55℃水浴中5分钟。 </li><li>在5分钟已经过去之后，取出管和擦拭用70％的乙醇。 </li><li>加入10升PI的双阳性和PI只有1.5毫升管。 </li><li>放置所有三个的K562靶细胞对照在孵化器在37℃下30分钟。 </li><li>后30分钟温育已经过去时，离心机所有三种的K562靶细胞对照为在163×g下2分钟。 </li><li>小心取出上清液，而不会干扰细胞沉淀。 </li><li>悬浮用新鲜的IMDM细胞培养基20μL的各控制，并在37℃培养箱，用5％ 的 CO 2离开至少30分钟最佳DIO信号。 注意:控制现在已经准备好通过成像流式细胞仪运行。 </li></ol> 4. NK细胞分离自动化<ol><li>打开电池用隔板，并允许启动周期来完成。 </li><li>确保所有液体的瓶子照明是绿色的，该废液瓶是空的。 </li><li>获得室温15毫升管架。 注意:选择是基于样品体积。例如，对于一个容积小于3毫升使用15毫升管架和用于体积超过3毫升用50毫升管架。 </li><li>标签样品管据此（每个样品/参与者重复）:参与者1全血样品;参与者1负分数;参与者1正分数。 </li><li>轻轻吸取1.5毫升的全血，从1.2步进入"全血样本"15毫升管。 </li><li>从步骤4.5将适当标记的15毫升"全血样品"管和标签15毫升"阴性部分"和从管架4.4步"阳性分数"管。使用以下几部件样品架设置:行（R1）A:全血标本，行（R2）B:阴性，未标记的部分，行（R4）C:正，磁标记部分。 </li><li>插入分隔架到MiniSampler为autolabeling。 </li><li>选择"试剂"的菜单上并突出，其中所述小瓶将被放置在隔板架的位置。 </li><li>选择"读取试剂"激活二维码阅读器。 </li><li>放置在从读码器盖0.5-2.5厘米之间的二维码阅读器的前部的适当的试剂瓶中。 注意:例如，对于NK细胞分离所需要的试剂是CD56。 </li><li>在二维码阅读器前最佳阅读的角度握住试剂瓶。 </li><li>将试剂瓶到适当的分离器机架位置。 </li><li>突出显示在屏幕上分离选项卡下的所需的位置。 </li><li>从标签子菜单，分配一个保姆tolabeling程序。 </li><li>分配细胞分离试剂CD56微珠机架位置1,2，3和4。 </li><li>选择"posselwb"离职计划。 </li><li>选择"冲洗"洗程序。 </li><li>将在"卷"子菜单使用数字键盘1500μL的样本量。 </li><li>选择键盘上的"Enter"选项。 </li><li>选择"运行"，启动细胞分离。 </li><li>选择"确定"，以确认有足够的缓冲区可用于处理所有样本。 </li></ol> 5. NK细胞计数随着细胞分离<ol><li>紧随与细胞分离细胞分离，检索阳性部分。留在室温下。这个级分含有所需纯的NK细胞群。 </li><li> 对于每个单独的样本，执行使用血球按照步骤2.2中的细胞计数。 <ol><li>计算后，记录细胞计数。 </li&gt; </ol></li></ol> 6.细胞毒性测定样品的制备<ol><li>准备和相应的标签1.5 mL管每个样品/参与者。 </li><li>吸管所需NK细胞和DIO-标有K562细胞比到每个管中。 注意:例如，K562靶细胞和NK效应细胞的所需的比例为1:5。 </li><li>离心机在135×g下5分钟。 </li><li>小心取出上清液，而不会干扰细胞沉淀。 </li><li>重悬的NK-DIO标记的K562细胞混合物中的NK细胞培养基的500微升无白细胞介素-2（IL-2）和2-巯基乙醇（2-ME）（不完全的NK细胞培养基）。 注:不完全的NK细胞培养基是最低基本培养基Eagle用碳酸氢钠，不含L-谷氨酰胺，核糖核苷和脱氧核糖核苷。 </li><li>添加5μL的PI的每管。 </li><li>离心机在163×g下2分钟。 </li><li>孵育细胞在37℃下2小时。 </li><li>继2小时培养，centrifuGE在163×g的2分钟。 </li><li>小心取出上清液，而不会干扰细胞沉淀。 </li><li>悬浮细胞在25μL的不完整的NK细胞培养基。 </li></ol> 7.准备自发的（"S"）样本<ol><li>吸移管将500μlDIO标记K562细胞到1.5mL管中（1×10 6个细胞/ mL的浓度）。 </li><li>加的PI的10微升每管。 </li><li>离心管在163×g下2分钟。 </li><li>孵育细胞在37℃下2小时。 </li><li>继2小时培养，离心机在163×g的2分钟。 </li><li>小心取出上清液，而不会干扰细胞沉淀。 </li><li>悬浮细胞在25μl的不完整的α-最低必需培养基（α-MEM）的细胞培养介质。 </li></ol> 8.数据采集与成像流式细胞仪<ol><li>按成像前门内侧的绿色按钮，流式细胞仪，打开仪器。 </li><li>打开人与成像相关联的升计算机流式细胞仪。 </li><li>启动软件流式细胞仪成像流程。 </li><li>点击"启动"按钮，冲洗系统，并准备样品线。 </li><li>一旦"启动"完成后，关闭了"校准"窗口。 </li><li>分配渠道:在顶部左侧，为了给它们分配点击每个通道上。 </li><li>在右侧，点击散点图按钮创建4散点图:生最大像素_MC_6 VS Area_M06，原最大像素_MC_2 VS Area_M02，原最大像素_MC_5 VS Area_M05和FieldArea_M01 VS AspectRatio_M01。 </li><li> 开始先使用"双重利好"控制分析样品。 <ol><li>点击"加载"。 </li><li>将1.5 ml管从3.4步"双积极"样品3.9到样品装载器。 </li><li>在"放大"选项卡下面的40X目标。 </li><li>打开405毫瓦642毫瓦的激光。 </li><li>打开"明场"通道。 </li><li>点击"选择强度。" </li><li>基于"双阳性"对照样品，确定为405毫瓦的激光所需的强度，这样，探测器不会过载。 注意:例如，对于这个实验的最佳强度设定在11毫瓦。 </li></ol></li><li> 所需的设置，实现后，采集数据。 <ol><li>在"文件获取"选项卡，输入自定义的文件名的文本。选择保存数据文件（S）的文件夹。 </li><li>进入细胞的数目，以获得下一个为"收集"。通常，这个数字1000之间变化至10000。 </li><li>点击"采集"。 注意:一旦获取的细胞的期望数目时，数据文件自动保存先前选择的文件夹中。 </li></ol></li><li> 收购完成后，加载下一个对照样品-迪欧唯一控制。 </STRONG&gt; <ol><li>点击"加载"。 </li><li>将1.5 ml管的"DIO只是"样品放入样品装载器。 </li><li>请选中"放大"选项卡下的40倍的目标。 </li><li>离开405毫瓦的激光开启。 </li><li>关掉642毫瓦的激光和"明场"通道。 注意:现在，所需的设置已经实现，可以将数据获取。 </li><li>在"文件获取"选项卡，输入自定义的文件名的文本并选择保存数据文件（S）的文件夹。进入细胞的数目，以获得下一个为"收集"。通常此数目为1,000。 </li><li>点击"采集"。 注意:一旦获取单元的期望数量的数据文件自动保存先前选择的文件夹中。 </li></ol></li><li>重复步骤8.10，为"PI唯一的"对照样品。实验样品现已可供收集。 </li><li> 办理remaini纳克实验样品，其中包括"自发的"S"样本"，如下所示: <ol><li>请选中"放大"选项卡下的40倍的目标。 </li><li>接通405毫瓦和642毫瓦的激光。 </li><li>启用"明场"通道。 </li><li>点击"组强度。" </li><li>在"文件获取"选项卡，输入自定义的文件名的文本并选择保存数据文件（S）的文件夹。输入自定义文件名的文本。 </li><li>进入细胞的数量来获取旁边的"收集"。 </li><li>点击"采集"按钮。 注意:一旦获取单元的期望数量的数据文件自动保存先前选择的文件夹中。 </li></ol></li><li>重复步骤8.12对所有实验样品。 </li><li>所有的实验数据和文件已被收集后，点击"关闭"按钮，消毒系统。 </li></ol> 9. IMAGIN摹流式细胞仪样品分析<ol><li>打开流式细胞仪分析软件应用的摄像流动。 </li><li>在"文件"，打开一个实验.RIF文件。 </li><li>建立一个使用单一颜色.RIF文件（DIO-只控制，只有PI控制，在步骤8.10和8.11创建）选择一个新的矩阵在成像流式细胞仪软件补偿选项卡下的"创建一个新的矩阵"。 注:该软件将提示输入单色文件的选择和将它们合并以创建一个矩阵文件（.ctm文件扩展名），即要被选择应用的信道补偿。 </li><li> 通过使用该软件的"积木"功能创建点图。 <ol><li>创建一个点图（BrightFieldGradient_RMS VS频率）门聚焦细胞。调用门"焦点访谈"（ 图1A）。 </li><li>使用"焦点访谈"门，共创美好的场区的点积VS明场长宽比至G<html>吃了单细胞。调用门"单"（ 图1B）。 </li><li>使用"单次"门，创造Intensity_MC_Ch02的VS Intensity_MC_Ch5点图。使用此图来识别和门DIO阳性细胞只（目标，活的）和PI-DIO双阳性细胞（靶，死）（ 图1C）。 注:所有地块的步骤9.4.1，9.4.2所述，9.4.3可以通过软件的"积木"功能来创建。 </li></ol></li><li>点击散点图的统计功能来访问每个门的细胞数量。 </li><li>计算使用下面的公式自发样品中死目标和实验样品的百分比: 样品％的死目标=（#dead目标×100）/（＃活靶子+ #dead目标） </li><li>使用下列公式计算细胞毒性: ％细胞毒性= [（实验死自发死亡）/（100-自发性死亡）]×100 </li></ol>_content"FO: - together.within页保留="1"&gt; <img alt="图1" src="/files/ftp_upload/54779/54779fig1.jpg" /> 图1:代表直方图，散点图和图像细胞毒活性分析。 （ 一 ）重点细胞分析。 （B）单细胞分析。 （C）靶细胞染色分析。所有测定都使用连接到每个事件的图象制作。这可以在分析软件，只需点击图形上的事件进行访问。 （D）一种双峰事件的代表图像，表示凋亡NK细胞和活的K562靶。 CH01，明视。 CH02，DIO。 CH05，PI。 <a href="http://ecsource.jove.com/files/ftp_upload/54779/54779fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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A., Travers, P., Walport, M., Shlomchik, 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=.">.</a> Immunobiology: The Immune System in Health and Disease. 5th edn. , (2001).</li></ol>

The authors have nothing to disclose.

在这项研究中所描述的方法直接测量一个人的NK细胞响应于刺激的比活性（在此特定情况下，长时间运动）。通常情况下，NK细胞是从使用密度梯度或细胞用标记物的组合排序一个人的血液中分离。虽然这些方法被广泛使用，它们具有许多缺点:它们是耗时的，涉及多个操作，并且在很大程度上依赖于用户的。其结果是，过度的应力被放置在分离的NK细胞，这可以导致增加的可变性，从实验进行实验...

自然杀伤细胞是先天免疫系统的一个基本要素。而他们非常规，他们必须识别并通过细胞-细胞接触，而不事先活化1消除异常细胞的能力。因此，它们构成抵御感染的快速线。运动，尤其是剧烈的，已经显示出瞬时抑制免疫系统的2，3，4，5。 NK细胞是特别容易出现这种效果4，6，7，从而有效地创建增强疾病敏感性的一个窗口。因此，干预措施的研究之前，期间或剧烈运动后以减少其对NK细胞功能的影响的目的是为运动员后比赛福祉特别的兴趣。 8，在白血细胞室的约1％; 2）NK细胞对压力非常敏感，依靠不断的暴露在生理条件下保持在实验过程中可行，稳定;和3）的标准测定法来确定NK细胞细胞毒性，如聚蔗糖梯度9和释放测定法10中 ，是不可靠的和不一致的。人样品的固有变异性只会加重了这些问题。干预期间收集的新鲜人类样本是相当受管制，很难弄到，相对于动物的样品或永生细胞系的至少。这减少的机会，重复实验或增加参与者的研究队列达到显著的统计阈值。总的来说，这些问题的支持简化协议，允许FO的需要R 2均高吞吐量和人类样品中NK细胞裂解活性的高可靠性分析。 我们建立了缩短所需的时间，以确定，同时尽量减少暴露于外来因素从人全血分离和测试NK细胞的工作流。该方法优化了使用两个仪器，在基于磁细胞分选和流式细胞仪的成像流，和一个特定的化验-，优化的T:E比率为允许减少或NK细胞细胞毒性的增加的检测。

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			<td height="21" style="height:21px;width:371px;">K-562 lymphoblasts</td>
			<td style="width:183px;">ATCC</td>
			<td style="width:136px;">CCL-243</td>
			<td style="width:585px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Iscove&#39;s Modified Dulbecco&#39;s Media</td>
			<td style="width:183px;">ATCC</td>
			<td style="width:136px;">30-2005</td>
			<td>High glucose, with L-Glutamine, with HEPES, Sterile-filtered</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Alpha Minimum Essential medium</td>
			<td style="width:183px;">ATCC</td>
			<td style="width:136px;">CRL-2407</td>
			<td style="width:585px;">Without ribonucleosides and deoxyribonucleosides but with 2 mM L-glutamine and 1.5 g/L sodium bicarbonate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Trypan Blue Solution 0.4%</td>
			<td style="width:183px;">Amresco</td>
			<td style="width:136px;">K940-100ML</td>
			<td>Tissue culture grade</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Propridium Iodide Staining Solution</td>
			<td style="width:183px;">BD Pharmingen</td>
			<td style="width:136px;">51-66211E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Vybranto DiO cell-labeling solution</td>
			<td style="width:183px;">Vybranto</td>
			<td style="width:136px;">V-22886</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">autoMACS Pro Separator</td>
			<td style="width:183px;">Miltenyi Biotec</td>
			<td style="width:136px;">130-092-545</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">autoMACS Running Buffer</td>
			<td style="width:183px;">Miltenyi Biotec</td>
			<td style="width:136px;">130-091-221</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">autoMACS Washing Buffer</td>
			<td style="width:183px;">Miltenyi Biotec</td>
			<td style="width:136px;">130-092-987</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">autoMACS Columns</td>
			<td style="width:183px;">Miltenyi Biotec</td>
			<td style="width:136px;">130-021-101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Whole Blood CD56 MicroBeads, human</td>
			<td style="width:183px;">Miltenyi Biotec</td>
			<td style="width:136px;">130-090-875</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ImageStream X Mark II Imaging Flow Cytometer</td>
			<td style="width:183px;">EMD Millipore</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Speedbeads</td>
			<td style="width:183px;">Amnis Corporation</td>
			<td style="width:136px;">400030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.4-0.7% Hypochlorite (Sterilizer)</td>
			<td style="width:183px;">VWR</td>
			<td>JT9416-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Coulter Clenz</td>
			<td style="width:183px;">Beckman Coulter</td>
			<td>8546929</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70% Isopropanol (Debubbler)</td>
			<td style="width:183px;">EMD Millipore</td>
			<td style="width:136px;">1.3704</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-PBS (Sheath fluid)</td>
			<td style="width:183px;">EMD Millipore</td>
			<td style="width:136px;">BSS-1006-B (1X)</td>
			<td>No calcium or magnesium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">INSPIRE Software</td>
			<td style="width:183px;">EMD Millipore</td>
			<td style="width:136px;"></td>
			<td>Version Mark II, September 2013</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ideas Application Software</td>
			<td style="width:183px;">EMD Millipore</td>
			<td style="width:136px;"></td>
			<td>Version 6.1, July 2014</td>
		</tr>
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</table>

flow cytometric analysis of natural killer cell lytic activity in human whole blood

NK细胞细胞毒性是一种广泛使用的度量，以确定外部干预的对NK细胞功能的影响。然而，该测定的准确性和重复性可以认为不稳定的，或者是因为用户的错误，或者因为NK细胞的实验操纵的敏感性。为了消除这些问题，即它们减少到最低限度的工作流正式成立，这里提出。为了说明，我们获得的血液样品中，在不同的时间点，从流道（N = 4）被提交给运动的激烈比赛的。首先，NK细胞被同时确定，并通过CD56标记和基于磁分选分离，直接从全血和从少1毫升。排序后的NK细胞被删除任何试剂或封盖抗体。他们可以以每毫升血液建立一个准确的NK细胞数量进行计数。其次，分类NK细胞（细胞效应或E）可以与3,3&#39;- Diotade混合cyloxacarbocyanine高氯酸盐（DIO）标记的K562细胞（靶细胞或T）在一个测定最优1:5 T:E比率，并分析使用成像流式细胞仪，其允许每个事件的可视化和任何假阳性的消除或假阴性（如双峰或效应细胞）。该工作流可以在大约4小时内完成，并允许甚至与人类的样品时非常稳定的结果。空闲时，研究团队可以在人类受试者的测试几种实验的干预措施，并比较不同的几个时间点测量，而不影响数据的完整性。

NK细胞计数的测定 测定对全血中的NK细胞数重运行的效果，使用图2中描述的运动方案。血样的初始抽血后运动前拉伸，运动后即刻，运动后1.5小时，最后24小时和48小时。每全血毫升NK细胞的浓度为每个转轮（ 图3A）和平均（ 图3B）对每个时间点进行测量。 我们的研究结果（ 图3A...

Watch this Scientific Journal Video about Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood at JoVE.com

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

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 ...

flow-cytometric-analysis-natural-killer-cell-lytic-activity-human

Research

JoVE Journal

Immunology and Infection

12.3K 次观看.  North Carolina A&T State University, North Carolina Research Campus. 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.

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

Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has been in the study of immunological synapse formation, due to the ability of the glass-supported planar lipid bilayers to mimic the surface of a target cell while forming a horizontal interface. The recent advances in super-resolution imaging have further allowed scientists to better view the fine details of synapse structure. In this study, one of these advanced techniques, stimulated emission depletion (STED), is utilized to study the structure of natural killer (NK) cell synapses on the supported lipid bilayer. Provided herein is an easy-to-follow protocol detailing: how to prepare raw synthetic phospholipids for use in synthesizing glass-supported bilayers; how to determine how densely protein of a given concentration occupies the bilayer's attachment sites; how to construct a supported lipid bilayer containing antibodies against NK cell activating receptor CD16; and finally, how to image human NK cells on this bilayer using STED super-resolution microscopy, with a focus on distribution of perforin positive lytic granules and filamentous actin at NK synapses. Thus, combining the glass-supported planar lipid bilayer system with STED technique, we demonstrate the feasibility and application of this combined technique, as well as intracellular structures at NK immunological synapse with super-resolution.

We describe here a combination of the glass-supported lipid bilayer technique of forming immunological synapses with the super-resolution imaging technique of stimulated emission depletion (STED) microscopy. The goal of this protocol is to provide users with the instructions necessary to successfully carry out these two techniques.

的自然杀伤细胞免疫突触上的玻璃支承平面脂双层超分辨率成像

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has ...

科研

免疫与感染

Whole-animal Imaging and Flow Cytometric Techniques for Analysis of Antigen-specific CD8+ T Cell Responses after Nanoparticle Vaccination

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various nanoparticle systems have been engineered to mimic features of pathogens to improve antigen delivery to draining lymph nodes and increase antigen uptake by antigen-presenting cells, leading to new vaccine formulations optimized for induction of antigen-specific CD8+ T cell responses. In this article, we describe the synthesis of a &#8220;pathogen-mimicking&#8221; nanoparticle system, termed interbilayer-crosslinked multilamellar vesicles (ICMVs) that can serve as an effective vaccine carrier for co-delivery of subunit antigens and immunostimulatory agents and elicitation of potent cytotoxic CD8+ T lymphocyte (CTL) responses. We describe methods for characterizing hydrodynamic size and surface charge of vaccine nanoparticles with dynamic light scattering and zeta potential analyzer and present a confocal microscopy-based procedure to analyze nanoparticle-mediated antigen delivery to draining lymph nodes. Furthermore, we show a new bioluminescence whole-animal imaging technique utilizing adoptive transfer of luciferase-expressing, antigen-specific CD8+ T cells into recipient mice, followed by nanoparticle vaccination, which permits non-invasive interrogation of expansion and trafficking patterns of CTLs in real time. We also describe tetramer staining and flow cytometric analysis of peripheral blood mononuclear cells for longitudinal quantification of endogenous T cell responses in mice vaccinated with nanoparticles.

We describe whole-animal imaging and flow cytometry-based techniques for monitoring expansion of antigen-specific CD8+ T cells in response to immunization with nanoparticles in a murine model of vaccination.

对于纳米颗粒疫苗接种后的分析抗原特异性CD8 + T细胞应答整体动物成像和流式细胞技术

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

分离和流式细胞仪分析从缺血小鼠脑免疫细胞

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the &#946;-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of&#160;how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists&#8217; discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

分离和胶质瘤浸润外周血单个核细胞的流式细胞仪分析

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

A Simple Flow Cytometric Method to Measure Glucose Uptake and Glucose Transporter Expression for Monocyte Subpopulations in Whole Blood

Monocytes are innate immune cells that can be activated by pathogens and inflammation associated with certain chronic inflammatory diseases. Activation of monocytes induces effector functions and a concomitant shift from oxidative to glycolytic metabolism that is accompanied by increased glucose transporter expression. This increased glycolytic metabolism is also observed for trained immunity of monocytes, a form of innate immunological memory. Although in vitro protocols examining glucose transporter expression and glucose uptake by monocytes have been described, none have been examined by multi-parametric flow cytometry in whole blood. We describe a multi-parametric flow cytometric protocol for the measurement of fluorescent glucose analog 2-NBDG uptake in whole blood by total monocytes and the classical (CD14++CD16-), intermediate (CD14++CD16+) and non-classical (CD14+CD16++) monocyte subpopulations. This method can be used to examine glucose transporter expression and glucose uptake for total monocytes and monocyte subpopulations during homeostasis and inflammatory disease, and can be easily modified to examine glucose uptake for other leukocytes and leukocyte subpopulations within blood.

Monocytes are integral components of the human innate immune system that rely on glycolytic metabolism when activated. We describe a flow cytometry protocol to measure glucose transporter expression and glucose uptake by total monocytes and monocyte subpopulations in fresh whole blood.

一个简单的流式细胞仪法测量葡萄糖的摄取和葡萄糖转运表达单核细胞亚群的全血

Monocytes are innate immune cells that can be activated by pathogens and inflammation associated with certain chronic inflammatory diseases. Activation of ...

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 ...

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore essential for understanding pathogenicity of different pathogens including HIV. Gamma delta (GD) T cells are the prototype of &#39;unconventional&#39; T cells and represent a relatively small subset of T cells defined by their expression of heterodimeric T-cell receptors (TCRs) composed of gamma and delta chains. This sets them apart from the classical and much better known CD4+ helper T cells and CD8+ cytotoxic T cells that are defined by alpha-beta TCRs. GD T cells often show tissue-specific localization and are enriched in epithelium. GD T cells orchestrate immune responses in inflammation, tumor surveillance, infectious disease, and autoimmunity.
Here, we present a method to reproducibly isolate and analyze human endocervical intraepithelial GD T lymphocytes. We have used endocervical cytobrush samples from women participating in the Women&#39;s Interagency HIV Infection Study (WIHS). Knowledge about GD T cells interactions during conditions in which there is an insult to the vaginal mucosal could be applied to any clinical study in which mucosal vulnerability is addressed, including the development of vaginal microbicides.In addition, knowledge about mucosal GD T cell responses has potential for application of GD T cell-based immune therapy in treating infectious diseases.

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

人类宫颈的γδT细胞的分离和流式细胞仪分析

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can have pathological significance. An ideal method for examining alterations to monocytes is whole blood flow cytometry as the minimal handling of samples by this method limits artifactual cell activation. However, many different approaches are taken to gate the monocyte subsets leading to inconsistent identification of the subsets between studies. Here we demonstrate a method using whole blood flow cytometry to identify and characterize human monocyte subsets (classical, intermediate, and non-classical). We outline how to prepare the blood samples for flow cytometry, gate the subsets (ensure contaminating cells have been removed), and determine monocyte subset expression of surface markers &#8212; in this example M1 and M2 markers. This protocol can be extended to other studies that require a standard gating method for assessing monocyte subset proportions and monocyte subset expression of other functional markers.

Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory).

全血流式细胞术分析人单核细胞亚群的表征

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...

Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a myeloid-biased 39-antibody CyTOF panel to evaluate the hematopoietic system with a focus on the neutrophil-lineage cells by using this protocol. The CyTOF result was analyzed with an open-resource dimensional reduction algorithm, viSNE, and the data was presented to demonstrate the outcome of this protocol. We have discovered new neutrophil-lineage cell populations based on this protocol. This protocol of fresh whole BM preparation may be used for 1), CyTOF analysis to discover unidentified cell populations from whole BM, 2), investigating whole BM defects for patients with blood disorders such as leukemia, 3), assisting optimization of fluorescence-activated flow cytometry protocols that utilize fresh whole BM.

Here, we present a protocol to process fresh bone marrow (BM) isolated from mouse or human for high-dimensional mass cytometry (Cytometry by Time-Of-Flight, CyTOF) analysis of neutrophil-lineage cells.

全骨髓的制备,用于中性粒系细胞的大规模细胞测定分析

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a ...

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are cleared from the human lung alveoli by being phagocytosed by innate monocytes and/or neutrophils. This protocol offers a fast and reliable measurement of phagocytosis by flow cytometry using fluorescein isothiocyanate (FITC)-labeled conidia for co-incubation with human leukocytes and subsequent counterstaining with an anti-FITC antibody to allow discrimination of internalized and cell-adherent conidia. Major advantages of this protocol are its rapidness, the possibility to combine the assay with cytometric analysis of other cell markers of interest, the simultaneous analysis of monocytes and neutrophils from a single sample and its applicability to other cell wall-bearing fungi or bacteria. Determination of percentages of phagocytosing leukocytes provides a means to microbiologists for evaluating virulence of a pathogen or for comparing pathogen wildtypes and mutants as well as to immunologists for investigating human leukocyte capabilities to combat pathogens.