Name: measuring granulocyte and monocyte phagocytosis and oxidative burst activity in human blood
Uploaded: 2016-09-12T13:00:00.000+00:00
Duration: 11 min 29 s
Description: Watch this Scientific Journal Video about Measuring Granulocyte and Monocyte Phagocytosis and Oxidative Burst Activity in Human Blood at JoVE.com

作者在此过程中的优化过程中承认扎克树，约翰·卡西和林恩Cialdella鉴的协助。

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.检测制剂<ol><li>收集，准备，并组织程序指定的材料（请参考具体的材料/设备表）。 <ol><li>标号12×75毫米的管。 <ol><li>标签两管为每个参与者:一个管的吞噬作用测定法（标记该管与黑色墨水和一个"F"为异硫氰酸荧光素[FITC]）和用于转播活性测定一个管（标记该管与红色墨水和一个" H"为二氢乙啶（他））。 注意:使用无菌12×75毫米管的建议，以减少意外的细胞活化并在背景噪声相关联的增加。 </li><li>标签三管的检测控制:一管的吞噬试验（FITC控制:标签此管BLACķ墨水和一个"F"），一个管的转播活性测定（HE控制:标示该管子用红墨水和一个"H"），而对于阴性对照一个管（血液只:标记该管与绿色油墨和"血止"）。 </li></ol></li><li>至少测定当天前一天制备储备溶液。 <ol><li>制备无菌磷酸盐缓冲盐水（PBS）。稀释百毫升10X PBS与900毫升18.2MΩH 2 O的过滤器具有0.2微米的过滤消毒。储存在4℃直至使用。 </li><li>制备无菌PBS葡萄糖。溶解1.0克葡萄糖的100毫升PBS中。过滤器具有0.2微米的过滤消毒。储存在4℃直至使用。 </li><li>制备无菌的0.1M柠檬酸盐缓冲液。溶解将1.2g柠檬酸和100毫升18.2兆欧H 2 O 1.1克柠檬酸钠的将pH调节至4.0。过滤器具有0.2微米的过滤消毒。储存在4℃直至使用。 </li><li>制备何原液。 - [Resuspend在1.0毫升二甲亚砜（DMSO）1.0毫克的HE。轻轻混匀，分装，并储存在-20°C，直到使用。 </li><li>制备FITC标记的金黄色葡萄球菌 （ 金黄色葡萄球菌 ）原液（2×10 9个粒子/ ml）。悬浮10毫克FITC-标记S的金黄色葡萄球菌在PBS 1.5毫升（或适量）。根据制造商的建议，分为三个500微升等份和超声。分装和储存在-20°C，直到使用。 </li><li>准备未标记S.葡萄球菌原液（2×10 9个粒子/ ml）。悬浮100毫克未标记的S.金黄色葡萄球菌在PBS中在15毫升（或适量）。根据制造商的建议，划分为三十500微升等份和超声。分装和储存在-20°C，直到使用。 </li></ol></li><li>在测定当天制备新鲜的工作的解决方案。 <ol><li>制备贺工作溶液。将10微升解冻HE原液990微升PBS葡萄糖。避光并保持在冰上直到使用。 </li><li>制备FITC标记S.金黄色葡萄球菌工作液（133333个/微升）。转移70微升解冻FITC标记S的金黄色葡萄球菌原液980微升的PBS。避光并保持在冰上直到使用。 </li><li>准备未标记S.金黄色葡萄球菌工作液（133333个/微升）。转让70微升未标记解冻的S.金黄色葡萄球菌原液980微升的PBS。避光并保持在冰上直到使用。 </li><li>准备淬灭溶液。添加750微升的0.4％台盼蓝溶液11.25毫升无菌0.1M柠檬酸缓冲液中。保持在冰 - 水浴直至使用。 </li></ol></li><li>有一个认证抽血根据世界卫生组织的抽血准则抽血。 <ol><li>抽血到含有K 2 EDTA 14毫升采血管。在根据制造商的说明VERT血液收集管。保持采血管在室温下在台式摇杆直至使用。使用这种血液在步骤1.2.1中描述的全血细胞计数（CBC）与白细胞（WBC）差异分析。 </li><li>抽取血液到含肝素锂14毫升采血管。根据制造商的说明倒置血液收集管。保持血液收集管在RT上的台式摇杆直到使用。使用这种血液在步骤2中描述的单核细胞和粒细胞吞噬功能和OB活性测定。 </li></ol></li></ol></li><li>确定将需要完成每个样品测定细菌的量（ 即 金黄色葡萄球菌 ）。 <ol><li>使用血液分析仪作为在制造商的说明中描述的血液与WBC差分分析进行CBC。记下白细胞计数的（细胞/毫升），中性粒细胞％和％单核细胞值。 </li><li>使用下面的方程来确定每个样本的嗜中性粒细胞和单核细胞计数。 中性粒细胞计数（细胞/毫升）=％中性粒细胞×白细胞（在细胞/ ml） 单核细胞计数（细胞/毫升）=％的单核细胞×白细胞（在细胞/ ml） </li><li>使用下面的方程来确定每个样本的巨噬细胞数量和存在于100微升样品的吞噬细胞的数量。 吞噬细胞计数（细胞/毫升）=中性粒细胞计数（细胞/毫升）+单核细胞计数（细胞/ ml）的 在100μl=吞噬细胞的数目（吞噬细胞计数（细胞/毫升））/ 10 </li><li>使用下面的公式来确定细菌（ 即 金黄色葡萄球菌 ）所需的各样品的数目和FITC标记S的量金黄色葡萄球菌或无标签S.应加入到每个管葡萄球菌工作溶液（133333粒子/ ml）。 注:目标细菌:吞噬细胞比例为8:1。 需要的细菌数量=吞噬细胞在100μl×8数 需要（在微升）FITC或未标记工作溶液的体积=（所需的细菌数）/（133333颗粒/微升） </li></ol></li></ol> 2.执行分析<ol><li>准备全血化验。 <ol><li>每个参与者和控制管，使用一个扩展长度枪头从肝素锂采血管转移100μl的全血，以适当标记的12×75毫米管的底部。更换帽。 注意:小心，以避免对12×75毫米管的两侧获得血液。 </li><li>使用微量添加何工作溶液到标记有红色墨水和一个"H"，包括何控制管的管10微升。更换帽和旋涡简要介绍。 </li><li>孵育在37℃水浴中的所有管道15分钟。然后，所有的试管立即传送到冰 - 水浴12分钟。 注:使用一个开放的金属架并轻轻摇动机架每隔5分钟，以确保该管是快速和充分地加热或冷却。 </li></ol></li><li>加细菌（ 即 金黄色葡萄球菌 ）的样品和对照管中。 <ol><li>使用微量添加适量未标记S的（步骤1.2.4计算） 葡萄球菌工作溶液到标记有红色墨水和一个"H"，包括何控制管的管中。更换帽和旋涡简要介绍。 </li><li>使用微量添加适量FITC标记S的（步骤1.2.4计算） 葡萄球菌工作溶液到标有黑色油墨和一个"F"，包括FITC控制管的管中。更换帽和旋涡简要介绍。 </li><li>涡"血只有"控制管，但是我也不会类型的细菌（ 如 金黄色葡萄球菌 ）添加到它。 </li><li>孵育在37℃水浴中的所有管道20分钟。使用一个开放的金属架，并轻轻摇动机架每5分钟以确保将管快速和充分地加热。培养结束后，立即将所有管转移到冰水浴中1分钟。 </li></ol></li><li>洗涤细胞。 <ol><li>使用一个转发器移液管至100μl的冰冷急冷溶液添加到所有的管。简要地更换盖，涡旋，然后在冰上孵育所有试管1分钟。 </li><li>使用一个转发器移液管到1ml冰冷的PBS添加到所有的管。简要地涡旋，然后添加另外2毫升冰冷的PBS对所有管和替换帽。离心机在4℃，5分钟和270 XG，然后所有管吸出上清液。 </li><li>重复步骤2.3.2一次（共2次洗涤）。 </li></ol></li><li>制备的样品进行流式细胞。 <ol><li>使用一个转发器移液管向50微升冰冷的胎​​牛血清添加到所有的管。简而言之振荡试管。 </li><li>传输所有管，以适当的旋转木马，并使用自动细胞裂解准备工作站红细胞CELL（RBC）裂解和WBC定影试剂以裂解RBC和如在制造商的说明确定的白细胞。 </li><li>自动化细胞裂解制备工作站运行完成后，包裹在铝箔和储存管在4℃冰箱中直到可以进行流式细胞术分析的流动。 </li></ol></li></ol> 3.流式细胞仪采集<ol><li>设置流式细胞仪系统。 <ol><li>预热流式细胞仪系统流中的指示由制造商提供的用户手册。然后，检查试剂和废物箱的水平。如果需要的话，填试剂槽和/或清空废液罐中。 </li><li>使用"清洁面板"功能来运行系统清洗周期。然后，运行对准和流体验证fluorospheres，并确保变异对于每个检测器的半峰值系数（HPCV）是在质量控制中所述的预定范围内（QC）协议由制造商提供河</li></ol></li><li>创建特定的检测，采集和仪器设置的协议。 <ol><li>创建吞噬功能和OB活性测定特异性检测收购协议。 注意:此处描述的采集协议可适于在流式细胞仪用蓝色激光（488纳米）的任何流动使用，FITC过滤器（530/30），以及何一个过滤器（二十六分之五百七十五）。 <ol><li>流式细胞仪打开系统采集软件的流程，并创建一个"新协议"。 </li><li>选择"FL1参数"为吞噬测定（FITC）和"FL2参数"为OB活性测定法（HE）。 </li><li>创建地块（ 即前向散射[FSC] 对侧向散射[SSC]点图，直方图FL1，FL2直方图）所需的收购。 </li><li>为WBC（白血球）创建多边形区域，对FSC 与 SSC的情节粒细胞和单核细胞人口和创建的FL1和FL2直方图线性区域。集＆＃34;停止计数"50000事件白细胞门。 </li><li>保存特异性检测，名称的协议。 </li></ol></li><li>创建吞噬功能和OB活性测定特异性检测的仪器设置的协议。 <ol><li>拖放从"资源管理器"中的"特定的检测，采集协议"（步骤3.2.1定义）的"收购管理器"中的流式细胞仪系统采集软件的流程。 注意:在这种方式添加的信息将总是出现在工作列表的末尾。 </li><li>输入样品信息到工作表，并保存工作表。 </li><li>从4℃冰箱中取出对照样品管，并允许他们平衡至室温15分钟。然后，短暂涡​​旋每个控制样品管。 </li><li>使用在步骤3.2.1中定义的"特定化验-采集协议"，以运行阴性对照样品（ 即 ，只有血液）和阳性对照样本（ 即何控制，FITC控制），通过流式细胞仪系统。查看直方图，并根据需要调整光电倍增管（PMT）的FITC和藻红蛋白（PE）通道的电压。 </li><li>涡对照样品，并根据在工作列表的顺序将它们转移至流式细胞系统转盘。然后，放置在采样室的旋转木马，并点击工具栏流式细胞仪"开始"按钮，获得的对照样品的数据。保存"XXX专业设置"设置协议。 </li></ol></li></ol></li><li>收购流式细胞仪系统的研究样本。 <ol><li>除去从4℃冰箱内的研究样品管，并允许平衡至室温15分钟。 </li><li>虽然样本平衡，拖动预定义的具体研究，收购协议的工作表空间，使项目工作列表。 </li><li>确认该"设置协议"被链接到与＃34;在步骤3.2.2中定义的特定试验设定的协议"，然后，输入身份信息（ 如姓名的研究，得出数，访问数，主题ID），每个管进入工作清单。 </li><li>在15分钟的平衡期，涡流研究样品后，并根据上工作列表的顺序将它们转移至流式细胞系统转盘。然后，放置在采样室的旋转木马，并点击工具栏流式细胞仪"开始"按钮，获得的研究样本数据。 </li><li>通过检查红细胞裂解效率和WBC细胞亚群的分布，比例，绝对计数和荧光强度每个样本的报告面板上查看数据的质量。 注:数据质量被认为是，如果所有的这些标准是其预定的限度内可以接受的。如果所有不符合标准，样品应重新处理。 </li><li>清理并关闭流式细胞仪系统。存储流式细胞仪在两个不同的电脑驱动器的数据，以防止意外的数据丢失。 </li></ol></li></ol> 4.数据分析<ol><li>执行吞噬分析。 <ol><li>整合所有的吞噬作用（ 即 F-标记）样品和控制列表模式数据（LMD）数据文件到一个单独的文件夹中。然后，打开仪分析软件的流动和将文件拖放到样本空间。 </li><li>设置白细胞，粒细胞，单核细胞，淋巴细胞和城门。 <ol><li>文件名双击打开任何"FITC对照样品"的。使用下拉菜单来设定x轴，以"FSC"和"直链"和y轴为"SSC"和"直链"。 </li><li>使用"多边形栅极选择"以制备总WBC种群的栅极。 注意:一定要包括所有的细胞在图表的边缘。标签此门"井喷"。 </li><li>关于"新的WBC门"双击并使用滴下拉菜单来设定x轴，以"FSC"和"线性"和y轴为"SSC"和"线性"。 </li><li>使用"多边形门选择"设置"粒门"和单核细胞门"，然后使用"椭圆形门选择"设置"淋巴细胞门"，因此标注每个门。拖动"闸门数据（％） "关闭的一侧，以使细胞很容易被观看。 </li></ol></li><li>设置"吞噬正栅极"和"吞噬负栅"为粒细胞和单核细胞群。 <ol><li>在步骤4.1.2.1用"FITC对照样品"双击。然后，点击"粒门"，并使用下拉菜单设置X轴为"FL1"和"日志"和y轴为"SSC"和"线性"。 </li><li>使用"正方形栅极选择"设定"吞噬负栅"和一个"吞噬POSIT香港专业教育学院门"。 注:由于FITC对照样品未接受细菌（ 即 金黄色葡萄球菌 ）的刺激，所有的细胞的理论上应该是吞噬负。建立这些人群的界限时，使用自由裁量权。 </li><li>重复步骤4.1.3.1和单核细胞人口步骤4.1.3.2。 </li></ol></li><li>添加统计"白细胞％"，"荧光强度"和"％吞噬阳性细胞"为粒细胞和单核细胞群。 <ol><li>点击步骤4.1.2.1用"FITC对照样品"。突出"粒门"，然后点击"工作区"和"添加统计"。突出"几何平均值"和"FL1日志"，然后单击"添加"。然后选择了"家长的频率"统计，然后单击"添加"。 注意:这会给整个人口粒的荧光强度（几何平均值）和白细胞（父）是粒细胞的百分比。 </li><li>点击步骤4.1.2.1用"FITC对照样品"。突出粒人口"吞噬积极门"，然后点击"工作区"和"添加统计"。突出"几何平均值"和"FL1日志"，然后单击"添加"。然后选择"家长的频率"统计，然后单击"添加"。 注意:这将得到吞噬阳性粒细胞的荧光强度（几何平均值），并是吞噬阳性粒细胞（父）的百分比。 </li><li>重复步骤4.1.4.1和单核细胞人口步骤4.1.4.2。 </li><li>点击步骤4.1.2.1用"FITC对照样品"。突出"淋巴细胞门"，然后点击"工作区"和"添加统计"。突出"家长的频率"统计，然后单击"添加"。 注:此会给WBC（父），这些淋巴细胞的百分比。 </li><li>突出显示所有步骤4.1.2.1使用FITC对照样品中创建了盖茨夫妇和统计数据。在屏幕顶部的"集团"面板将它们拖动到"全样本"。 注意:这将应用这些设置应用于所有的研究样本。 </li><li>保存和命名工作作为包含LMD文件夹中的同一个文件夹.wsp文件。 </li></ol></li><li>调整单个样本门。 <ol><li>点击在数据集中的"第一个样本"，并确认WBC门适当配合屏幕上的细胞分布。如果没有，请单击并拖动门的边缘调整该样品的大门。点击"右箭头"（即"˃"）以前进到下一个样本。重复此步骤，直到所有的样品进行了审查。 </li><li>点击"WBC门"在数据集中的第一个样本，确认"大ulocyte门"，"单核细胞门"和"淋巴细胞门"对每个样品适当地适应屏幕上的细胞分布。根据需要进行调整。点击"右箭头"（即 "˃"）以前进到下一个样本。 </li><li>重复步骤4.1.5.2直到所有样品都进行了审查。然后，点击"保存"。 </li></ol></li><li>在电子表格程序显示的数据。 <ol><li>点击"表格编辑器"图标，在屏幕的顶部。点击"编辑"，"关键词"和"$ DATE"，包括对最终电子表格中的采样日期。然后，单击"编辑"，"关键词"和"@ SAMPLEID1"，包括对最终电子表格中的抽样鉴定。重复"@ SAMPLEID2"，"@ SAMPLEID3"和"@ SAMPLEID4"。 </li><li>调整"表编辑器"屏幕和"样品"屏幕，这样他们既可以在电脑屏幕上观看。塞莱CT样品表上的"样品"。然后，将每个感兴趣的统计数据的"表编辑器"。 <ol><li>在"频率。家长"的"粒门"中列出统计拖动到"表编辑器"。键入"％WBC粒细胞"中的"表编辑器"的"名称"列下。 </li><li>在"粒门"中列出的"几何平均数"统计拖到"表编辑器"。键入"几何平均荧光，粒"中的"表编辑器"的"名称"列下。 </li><li>在"频率。家长"的"粒/吞噬正门"所列出的统计拖动到"表编辑器"。键入"％正吞噬粒"中的"表编辑器"的"名称"列下。 </li><li>拖动"粒/正吞噬门"中列出的"几何平均数"统计为thE"表编辑器"。键入"几何平均荧光，正吞噬粒"中的"表编辑器"的"名称"列下。 </li><li>在"频率。家长"的"单核细胞门"中列出统计拖动到"表编辑器"。键入"％WBC单核细胞"中的"表编辑器"的"名称"列下。 </li><li>在"单核细胞门"中列出的"几何平均数"统计拖到"表编辑器"。键入"几何平均荧光，单核细胞"中的"表编辑器"的"名称"列下。 </li><li>在"频率。家长"的"单核/吞噬正门"所列出的统计拖动到"表编辑器"。键入"表编辑器"的"名称"列下的"吞噬％阳性单核细胞"。 </li><li>拖动下的"单核/ Phagocy列出的"几何平均数"统计细胞增多正门"到"表编辑器"，键入"几何平均荧光，吞噬阳性单核细胞表编辑器的"姓名"栏"，"下"。 </li><li>在"频率。家长"的"淋巴细胞门"中列出统计拖动到"表编辑器"。键入"％WBC淋巴细胞"中的"表编辑器"的"名称"列下。 </li></ol></li><li>拖放在"表编辑器"中可见的参数，直到他们被安排在优先顺序。 </li><li>点击"保存"。然后，单击"创建表"以电子表格格式来显示数据。点击"另存为"，保存并命名为工作.xlsx文件。 </li></ol></li></ol></li><li>对于OB活性测定重复步骤4.1（ 即 H标识）样品和对照LMD数据文件。 注意:一定要的所有引用替换吞噬一起OB活动引用。 </li></o升&gt;

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The authors have nothing to disclose.

这份手稿提供了一个一步一步的协议粒细胞和单核细胞功能的两个指标的决心。我们已经确定了对这个实验成功的关键几步之遥。一个这样的步骤是发生之前立即加入细菌的冰浴温育。所有样品的彻底冷却将最大限度地减少温度对吞噬和OB活性的作用。另一个关键步骤是在加入细菌到各样品中。 A 8:1的细菌:巨噬细胞产生比最佳效果;然而其他研究人员可能会发现，这个比例将需要进行修改，以产生...

粒和单核细胞的吞噬作用/氧化爆发（OB）活性测定是经常用来收集关于以下长时间和密集运动先天性免疫功能信息的简单，直接的技术。1-4当研究免疫系统的反应一种刺激，粒细胞和单核细胞是特别感兴趣的，因为它们在宿主发挥防御核心作用，并且是第一个免疫细胞在感染部位蓄积。5嗜中性粒细胞，一式粒，是第一细胞移位到受损肌肉组织下面的剧烈运动。6吞噬和OB活性的粒细胞和单核细胞的功能，7的最常见的措施和优选作为因为它们在两个感染过程和修复过程中心作用先天免疫细胞功能的指标。 在这个手稿utili描述的测定ZES一个基本的流式细胞仪提供全血粒细胞和单核细胞吞噬功能和OB活动的定量测定。在此技术中使用的全血是有利的，因为它允许在不一项费时的纯化方法来进行测定。该测定可以很容易地进行修改，以适应各种研究设计和实验室能力。例如，Liao等利用类似的技术，以显示该嗜中性粒细胞的OB活性增加和中性粒细胞的吞噬作用降低创伤性脑损伤。8在另一实例中，麦克法林等人描述该技术，使用别藻蓝蛋白的优雅修改（APC ）结合的CD66b和高性能串联APC标记的CD45标记与基于图像的流式细胞仪在其吞噬作用和OB活动方面分类粒细胞。-7,9- 我们的研究小组已用这种TE的各种改编chnique如超重，肥胖和运动人群先天免疫功能，为近20年来的一个指标。10,11文字下面将提供一步一步的指导读者进行这种测定，并强调指出，读者可以适应领域以满足他们的研究的需要。

<table><tbody><tr><td>12 x 75 mm tubes, with cap</td><td>VWR</td><td>20170-579</td><td>You will need 2 tubes per sample (&quot;H&quot; and &quot;F&quot;) plus 3 tubes per batch (for assay controls; &quot;F&quot;, &quot;H&quot;, and &quot;Blood only&quot;).</td></tr><tr><td>PBS (10x), pH 7.4</td><td>Life Technologies</td><td>70011-044</td><td></td></tr><tr><td>Bottle top 0.2 &amp;micro;m cellulose acetate filter (500 ml capacity)</td><td>Fisher Scientific</td><td>09-741-07&amp;nbsp;</td><td></td></tr><tr><td>Glucose, powder</td><td>Life Technologies</td><td>15023-021</td><td></td></tr><tr><td>Citric acid (anhydrous, cell culture tested, plant cell culture tested)</td><td>Sigma-Aldrich</td><td>C2404-100G</td><td></td></tr><tr><td>Sodium citrate tribasic dihydrate</td><td>Sigma-Aldrich</td><td>S4641-25G</td><td></td></tr><tr><td>Dihydroethidium (Hydroethidine) (HE)</td><td>Life Technologies</td><td>D11347</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO) Anhydrous</td><td>Life Technologies</td><td>D12345</td><td></td></tr><tr><td>Staphylococcus aureus&amp;nbsp;(Wood strain without protein A) BioParticles, fluorescein conjugate (FITC)</td><td>Life Technologies</td><td>S2851</td><td></td></tr><tr><td>Staphylococcus aureus&amp;nbsp;(Wood strain without protein A) BioParticles, unlabeled</td><td>Life Technologies</td><td>S-2859</td><td></td></tr><tr><td>Trypan Blue Solution, 0.4%</td><td>Life Technologies</td><td>15250-061</td><td></td></tr><tr><td>4.0 ml vacutainer containing 7.2 mg K&lt;sub&gt;2&lt;/sub&gt;EDTA, spray-dried</td><td>VWR</td><td>BD367861</td><td>You will need 1 K&lt;sub&gt;2&lt;/sub&gt;EDTA blood collection tube per sample.</td></tr><tr><td>4.0 ml vacutainer containing 68 USP units Lithium Heparin, spray-coated</td><td>VWR</td><td>BD367884</td><td>You will need 1 Lithium Heparin blood collection tube per sample.</td></tr><tr><td>COULTER Ac&amp;middot;T 5diff CP</td><td>Beckman Coulter</td><td>6605705</td><td>&lt;em&gt;i.e.&lt;/em&gt;, hematology analyzer</td></tr><tr><td>COULTER Ac&amp;middot;T 5diff Rinse</td><td>Beckman Coulter</td><td>8547167</td><td></td></tr><tr><td>COULTER Ac&amp;middot;T 5diff Fix</td><td>Beckman Coulter</td><td>8547171</td><td></td></tr><tr><td>COULTER Ac&amp;middot;T 5diff WBC Lyse</td><td>Beckman Coulter</td><td>8547170</td><td></td></tr><tr><td>COULTER Ac&amp;middot;T 5diff Hgb Lyse</td><td>Beckman Coulter</td><td>8547168</td><td></td></tr><tr><td>COULTER Ac&amp;middot;T 5diff Cal Calibrator</td><td>Beckman Coulter</td><td>7547175</td><td></td></tr><tr><td>COULTER Ac&amp;middot;T 5diff Control Plus</td><td>Beckman Coulter</td><td>7547198</td><td></td></tr><tr><td>AcT 5diff Diluent</td><td>Beckman Coulter</td><td>8547169</td><td></td></tr><tr><td>200 &amp;micro;l extended-length pipette tips</td><td>VWR</td><td>37001-526</td><td></td></tr><tr><td>Insulated ice pan</td><td>VWR</td><td>89198-980</td><td>For ice water bath.</td></tr><tr><td>Open metal tube rack</td><td>VWR</td><td>60916-702</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Life Technologies</td><td>26140-111</td><td></td></tr><tr><td>TQ-Prep Workstation</td><td>Beckman Coulter</td><td>6605429</td><td>&lt;em&gt;i.e.&lt;/em&gt;, automated cell lyse preparation workstation</td></tr><tr><td>ImmunoPrep Reagent System</td><td>Beckman Coulter</td><td>7546999</td><td>&lt;em&gt;i.e.&lt;/em&gt;, RBC lyse/WBC fix reagent system</td></tr><tr><td>Cytomics FC500 MCL Flow Cytometry System with CXP Software</td><td>Beckman Coulter</td><td>626553</td><td></td></tr><tr><td>IsoFlow Sheath Fluid</td><td>Beckman Coulter</td><td>8547008</td><td></td></tr><tr><td>COULTER CLENZ</td><td>Beckman Coulter</td><td>8546929</td><td></td></tr><tr><td>Flow-Check Fluorospheres&amp;nbsp;</td><td>Beckman Coulter</td><td>6605359</td><td>&lt;em&gt;i.e.&lt;/em&gt;, alignment and fluidics verification fluorospheres</td></tr><tr><td>FlowJo Software</td><td>FlowJo</td><td>FlowJo</td><td>&lt;em&gt;i.e.&lt;/em&gt;, flow cytometry analysis software</td></tr><tr><td>Excel Software</td><td>Microsoft</td><td>Microsoft Excel</td><td>&lt;em&gt;i.e.&lt;/em&gt;, electronic spreadsheet program</td></tr></tbody></table>

measuring granulocyte and monocyte phagocytosis and oxidative burst activity in human blood

The granulocyte and monocyte phagocytosis and oxidative burst (OB) activity assay can be used to study the innate immune system. This manuscript provides the necessary methodology to add this assay to an exercise immunology arsenal. The first step in this assay is to prepare two aliquots ("H" and "F") of whole blood (heparin). Then, dihydroethidium is added to the H aliquot, and both aliquots are incubated in a warm water bath followed by a cold water bath. Next, Staphylococcus aureus (S. aureus) is added to the H aliquot and fluorescein isothiocyanate-labeled S. aureus is added to the F aliquot (bacteria:phagocyte = 8:1), and both aliquots are incubated in a warm water bath followed by a cold water bath. Then, trypan blue is added to each aliquot to quench extracellular fluorescence, and the cells are washed with phosphate-buffered saline. Next, the red blood cells are lysed, and the white blood cells are fixed. Finally, a flow cytometer and appropriate analysis software are used to measure granulocyte and monocyte phagocytosis and OB activity. This assay has been used for over 20 years. After heavy and prolonged exertion, athletes experience a significant but transient increase in phagocytosis and an extended decrease in OB activity. The post-exercise increase in phagocytosis is correlated with inflammation. In contrast to normal weight individuals, granulocyte and monocyte phagocytosis is chronically elevated in overweight and obese participants, and is modestly correlated with C-reactive protein. In summary, this flow cytometry-based assay measures the phagocytosis and OB activity of phagocytes and can be used as an additional measure of exercise- and obesity-induced inflammation.

长期和剧烈运动，对先天免疫功能产生深远的影响，包括自然杀伤细胞的功能，对病毒感染的巨噬细胞因子介导的反应，和粒细胞和单核细胞吞噬功能和OB活动。多项研究表明显著的粒细胞和单核细胞吞噬功能的增加，运动后，反映了肌肉损伤和代谢的需求引起的炎症。与此相反，粒细胞和单核OB活性降低运动后，可能是易感性增加感染的一种免疫指标。例如， 图1</...

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Measuring Granulocyte and Monocyte Phagocytosis and Oxidative Burst Activity in Human Blood

The purpose of this manuscript is to present a method for measuring monocyte and granulocyte phagocytosis and oxidative burst activity in human blood samples.

人体血液中测粒及单核细胞吞噬功能和呼吸爆发活动

The granulocyte and monocyte phagocytosis and oxidative burst (OB) activity assay can be used to study the innate immune system. This manuscript provides ...

measuring-granulocyte-monocyte-phagocytosis-oxidative-burst-activity

Research

JoVE Journal

Immunology and Infection

14.1K 次观看.  Appalachian State University. The purpose of this manuscript is to present a method for measuring monocyte and granulocyte phagocytosis and oxidative burst activity in human blood samples.

视频: Measuring Granulocyte and Monocyte Phagocytosis and Oxidative Burst Activity in Human Blood

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the phenotypic and functional differences between murine and human monocyte-derived macrophages. Therefore, we here describe an in vitro model to generate and study primary human macrophages. Briefly, after density gradient centrifugation of peripheral blood drawn from a forearm vein, monocytes are isolated from peripheral blood mononuclear cells using negative magnetic bead isolation. These monocytes are then cultured for six days under specific conditions to induce different types of macrophage differentiation or polarization. The model is easy to use and circumvents the problems caused by species-specific differences between mouse and man. Furthermore, it is closer to the in vivo conditions than the use of immortalized cell lines. In conclusion, the model described here is suitable to study macrophage biology, identify disease mechanisms and novel therapeutic targets. Even though not fully replacing experiments with animals or human tissues obtained post mortem, the model described here allows identification and validation of disease mechanisms and therapeutic targets that may be highly relevant to various human diseases.

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages are important cells of the innate immune system. Here, we describe an easy to use in vitro model to generate these cells. Using gradient centrifugation, negative bead isolation and specific cell culture conditions, monocyte-derived macrophages can be generated for phenotypic and functional studies.

一个<em在体外</em&gt;模型研究人类巨噬细胞分化和极化的异质性

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

科研

免疫与感染

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 ...

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

Granulocytes play a key role in the body&#8217;s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte function, in general these are limited in terms of the information they can provide. For example, most existing assays merely provide a percentage of how many granulocytes are activated following a single, fixed length incubation. Complicating matters, most assays focus on only one aspect of function due to limitations in detection technology. This report demonstrates a technique for simultaneous measurement of granulocyte phagocytosis of bacteria and oxidative burst. By measuring both of these functions at the same time, three unique phenotypes of activated granulocytes were identified: 1) Low Activation (minimal phagocytosis, no oxidative burst), 2) Moderate Activation (moderate phagocytosis, some oxidative burst, but no co-localization of the two functional events), and 3) High Activation (high phagocytosis, high oxidative burst, co-localization of phagocytosis and oxidative burst). A fourth population that consisted of inactivated granulocytes was also identified. Using assay incubations of 10, 20, and 40-min the effect of assay incubation duration on the redistribution of activated granulocyte phenotypes was assessed. A fourth incubation was completed on ice as a control. By using serial time incubations, the assay may be able to able to detect how a treatment spatially affects granulocyte function. All samples were measured using an image-based flow cytometer equipped with a quantitative imaging (QI) option, autosampler, and multiple lasers (488, 642, and 785 nm).

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

This method demonstrates a technique for assessing granulocyte function by simultaneously measuring phagocytosis of bacteria and oxidative burst. Image-based flow cytometry allowed for the identification of three distinct subsets of activated granulocytes that all differed in their relative functional capacity.

基于图像的流式细胞仪技术来评估变化粒细胞功能<em&gt;在体外</em

Granulocytes play a key role in the body&#8217;s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte ...

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly versatile in vitro assay that can be modified to examine different aspects of antibody and Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis in both research and clinical settings. The assay utilizes adherent monocytes from peripheral blood mononuclear cells isolated from mammalian whole blood. MMA has been described for use in both human and murine investigations. These monocytes express Fc&#947;Rs (e.g., Fc&#947;RI, Fc&#947;RIIA, Fc&#947;RIIB, and Fc&#947;RIIIA) that are involved in immune responses. The MMA exploits the mechanism of Fc&#947;R-mediated interactions, phagocytosis in particular, where antibody-sensitized red blood cells (RBCs) adhere to and/or activate Fc&#947;Rs and are subsequently phagocytosed by the monocytes. In vivo, primarily tissue macrophages found in the spleen and liver carry out Fc&#947;R-mediated phagocytosis of antibody-opsonized RBCs, causing extravascular hemolysis. By evaluating the level of phagocytosis using the MMA, different aspects of the in vivo Fc&#947;R-mediated process can be investigated. Some applications of the MMA include predicting the clinical relevance of allo- or autoantibodies in a transfusion setting, assessing candidate drugs that promote or inhibit phagocytosis, and combining the assay with fluorescent microscopy or traditional Western immunoblotting to investigate the downstream signaling effects of Fc&#947;R-engaging drugs or antibodies. Some limitations include the laboriousness of this technique, which takes a full day from start to finish, and the requirement of research ethics approval in order to work with mammalian blood. However, with diligence and adequate training, the MMA results can be obtained within a 24-h turnover time.

The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis.

单核细胞单层试验中用于评估Fcγ受体介导的吞噬作用

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...

Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of the actin cytoskeleton to capture, envelop and engulf large particles. Although phagocytic receptors, downstream signaling pathways, and effectors, such as Rho GTPases, have been identified, the dynamic cytoskeletal remodeling of specific receptor-mediated phagocytic events remain unclear. Four decades ago, two distinct mechanisms of phagocytosis, exemplified by Fc&#947; receptor (Fc&#947;R)- and complement receptor (CR)-mediated phagocytosis, were identified using scanning electron microscopy. Binding of immunoglobulin G (IgG)-opsonized particles to Fc&#947;Rs triggers the protrusion of thin membrane extensions, which initially form a so-called phagocytic cup around the particle before it becomes completely enclosed and retracted into the cell. In contrast, complement opsonized particles appear to sink into the phagocyte following binding to complement receptors. These two modes of phagocytosis, phagocytic cup formation and sinking in, have become well established in the literature. However, the distinctions between the two modes have become blurred by reports that complement receptor-mediated phagocytosis may induce various membrane protrusions. With the availability of high resolution imaging techniques, phagocytosis assays are required that allow real-time 3D (three dimensional) visualization of how specific phagocytic receptors mediate the uptake of individual particles. More commonly used approaches for the study of phagocytosis, such as end-point assays, miss the opportunity to understand what is happening at the interface of particles and phagocytes. Here we describe phagocytic assays, using time-lapse spinning disk confocal microscopy, that allow 3D imaging of single phagocytic events. In addition, we describe assays to unambiguously image Fc&#947; receptor- or complement receptor-mediated phagocytosis.

Here we describe methods using spinning disk confocal microscopy to image single phagocytic events by mouse resident peritoneal macrophages. The protocols can be extended to other phagocytic cells.

小鼠巨噬细胞吞噬的延时3D 成像

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of ...

Assessing Respiratory Immune Responses to Haemophilus Influenzae

Haemophilus influenzae (Hi) is a prevalent bacterium found in a range of respiratory conditions. A variety of different assays/techniques may be used to assess the respiratory immune/inflammatory response to this bacterium. Flow cytometry and confocal microscopy are fluorescence-based technologies that allow detailed characterization of biological responses.&#160;Different forms of Hi antigen can be used, including cell wall components, killed/inactivated preparations, and live bacteria. Hi is a fastidious bacterium that requires enriched media but is generally easy to grow in standard laboratory settings. Tissue samples for stimulation with Hi may be obtained from peripheral blood, bronchoscopy, or resected lung (e.g., in patients undergoing surgery for the treatment of lung cancer). Macrophage and neutrophil function may be comprehensively assessed using flow cytometry with a variety of parameters measured, including phagocytosis, reactive oxygen species, and intracellular cytokine production. Lymphocyte function (e.g., T cell and NK cell function) may be specifically assessed using flow cytometry, principally for intracellular cytokine production. Hi infection is a potent inducer of extracellular trap production, both by neutrophils (NETs) and macrophages (METs). Confocal microscopy is arguably the most optimal way to assess NET and MET expression, which may also be used to assess protease activity. Lung immunity to Haemophilus influenzae can be assessed using flow cytometry and confocal microscopy.

Assessing Respiratory Immune Responses to Haemophilus Influenzae

Haemophilus influenzae induces inflammation in the respiratory tract. This article will focus on the use of flow cytometry and confocal microscopy to define immune responses by phagocytes and lymphocytes in response to this bacterium.

评估对流感嗜血杆菌的呼吸道免疫应答

Haemophilus influenzae (Hi) is a prevalent bacterium found in a range of respiratory conditions. A variety of different assays/techniques may be used to ...

Prediction of Red Blood Cell Antibody Significance Using the Monocyte-Macrophage Assay

Derived from monocytes in the bone marrow, macrophages are large, innate immune cells that play a major role in clearing dead cells, debris, tumor cells, and foreign pathogens. The phagocytic capacity of monocytes versus macrophages is a concept that is not well understood. Here, we aim to examine a difference in the phagocytosis of monocytes versus macrophages, specifically M1/M2 macrophages, against various opsonized red cells using a modified and updated version of the established monocyte monolayer assay (MMA). Peripheral blood mononuclear cells (PBMCs) were isolated from donor buffy coats. Using purified monocytes, inflammatory M1 and anti-inflammatory M2 macrophages were produced by in vitro culture and polarization. M1/M2 cells were harvested and used in an MMA-like assay, which we refer to as the M-MA, to decipher clinically significant phagocytosis of various red cell antibodies. A phagocytic index (PI) &#62; 5 was deemed clinically significant phagocytosis with the use of monocytes. A phagocytic index (PI) &#62; 12 was deemed clinically significant phagocytosis with the use of M1/M2 macrophages. M2 macrophages demonstrate an increased ability to phagocytose opsonized RBCs compared to monocytes and M1s. The same weak antibody (anti-S) yields significant phagocytosis with only M2 macrophages (PI=43) but not M1s (PI=2) or monocytes (PI=0), and this was demonstrated repeatedly using various antibodies. The use of M2 macrophages instead of monocytes may allow for more accurate results as these cells are more phagocytic, offering further clinical relevance to the assay. Further studies with different antibodies to red blood cells, including validation of the monocyte-macrophage assay (M-MA) with antibodies having known clinical significance, may show the M-MA&#160;more useful to help predict clinically significant red cell alloantibodies and transfusion reactions. This method will advance the field of transfusion medicine and immunology.

We have developed enhancements and updated methods for the existing monocyte-monolayer assay (MMA), in which macrophages are used to help better predict the clinical relevance of red cell alloantibodies in transfusion medicine and immunology. This assay is named the monocyte-macrophage assay (M-MA).

使用单核细胞-巨噬细胞测定预测红细胞抗体的意义

Derived from monocytes in the bone marrow, macrophages are large, innate immune cells that play a major role in clearing dead cells, debris, tumor cells, ...

Real-Time Imaging of Macrophage Phagocytosis of IgG-Opsonized Red Blood Cells

Source: Horsthemke, M., et al. <a href="https://app.jove.com/v/57566">Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages.</a> J. Vis. Exp. (2018)
This video demonstrates an assay to monitor independent macrophage-mediated red blood cell (RBC) phagocytosis events. Mouse immunoglobulin G (IgG)-opsonized human RBCs are incubated with mouse macrophages and observed under a confocal microscope. The RBCs are engulfed by macrophages via a phagocytic cup formation and targeted for lysosomal degradation.

IgG 调导红细胞巨噬细胞吞噬作用的实时成像

Source: Horsthemke, M., et al. Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages. J. Vis. Exp. (2018)
This video demonstrates an assay to monitor ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Specialized Assays

Quantitative Flow Cytometric Measurement of Fungal Spore Phagocytosis by Human Phagocytes

Source: Hartung, S., et al., <a href="https://app.jove.com/v/60397">Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry.</a> J. Vis. Exp. (2019)
This video demonstrates a method for quantifying the phagocytosis of FITC-labeled pathogenic fungal spores by human leukocytes. Phagocytosed spores are discerned from cell-adherent spores through counterstaining, followed by flow cytometric analysis.

人吞噬细胞真菌孢子吞噬作用的定量流式细胞术测量

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Phagocytosis Assay

	Incubate 2 x ...

An Assay to Assess Phagocytosis and Oxidative Burst Activity In Granulocytes and Monocytes

Source: Meaney, M. P. et al., <a href="https://app.jove.com/v/54264">Measuring Granulocyte and Monocyte Phagocytosis and Oxidative Burst Activity in Human Blood.</a> J. Vis. Exp. (2016)
This video demonstrates the assay to confirm phagocytic activity by cells in human blood via flow cytometry. The granulocytes and monocytes confirm their phagocytic activity by showing green and red fluorescence confirming the presence of engulfed bacteria and oxidative burst activity respectively.

评估粒细胞和单核细胞吞噬作用和氧化爆发活性的测定

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human ...