这项工作是由基金会因诺琴蒂基金会意大利语二TECNOLOGIA支持。 作者要感谢美天旎生物技术有限公司（贝尔吉施 - 格拉德巴赫，德国）这个手稿的赞助，保罗Petrucciani博士提供人体血沉棕黄层（免疫血液学和输血服务中心，医院洛蒂蓬泰代拉，意大利比萨部）和教授马西莫Pasqualetti（生物系 - 细胞单位与发育生物学，比萨，意大利比萨大学）住房鼠标殖民地。

操守准则以下卫生意大利教育部的指导方针人类和动物样品进行处理，在第116/92号和欧洲共同体理事会指令86/609/EEC。 1。单核细胞培养<ol><li>培养人THP-1细胞中的T-75培养瓶中含补充有10％人血清，50单位/ ml青霉素和50毫克/毫升链霉素，在5％CO 2 0.05 mM的β-巯基乙醇，在37℃的RPMI-1640培养基。 </li><li>分裂的文化融合时（每隔3-5天）。 </li></ol> 2。从巴菲大衣分离外周血单个核细胞（PBMC）中的<ol><li>启动隔离协议之前，准备的磷酸盐缓冲盐水（PBS）pH 7.2的溶液中，2 mM的乙二胺四乙酸（EDTA），0​​.5％牛血清白蛋白（BSA），加入5ml的BSA储备液至95毫升漂洗缓冲液（1： 20稀释）。脱气缓冲区和保持低温（2-8℃）。 请注意：未脱气缓冲，可能会导致在不到最佳的效果，因为气泡会阻塞隔离柱（见步骤3.2）。 注：抗凝枸橼酸葡萄糖式-A（ACD-A）或柠檬酸磷酸葡萄糖（CPD）可交替使用。相反，来自其他物种的其他蛋白白蛋白或血清可以代替BSA。不要使用含Ca 2 +或Mg 2 +缓冲区。 </li><li>从健康的捐赠者（年龄在20-60岁）获得棕黄色的大衣。 </li><li>制备50ml锥形管中用于密度梯度离心法。确定所需的处理血液（每管可处理35ml的稀释血液），并添加15毫升的Ficoll-Paque上的每个空管的管的数目。 </li><li>稀释加入8ml血液24毫升的PBS / BSA / EDTA溶液（1:4稀释）。 </li><li>仔细层（密度=1.077克/毫升）中的每个的50ml锥形管上的Ficoll-Paque上的顶部的稀释液。不要混合使用血液和菲可 - 帕克。 重要提示 ：为避免血液和菲可-帕克的混合，保持管在45°角，慢慢地一层血合剂。 </li><li>离心机在400×g离心30分钟，在4℃下单核细胞（MNCs）将保持在所述等离子的Ficoll-Paque上的接口，而粒细胞和红细胞的沉淀是由于较高密度的Ficoll-Paque上的渗透压。 </li><li>转移的间期细胞（外周血单核细胞，PBMC）中，以一个新的50ml管中在4℃下填充用PBS / BSA / EDTA和离心机在300×g离心10分钟</li><li>用PBS / BSA / EDTA两次洗涤沉淀，除去血小板。转速为200×g离心10分钟，在4°C。 </li><li>培养的细胞在完全RPMI-1640含10％人类血清和抗生素或进行单核细胞的纯化。 </li></ol> 3。从外周血单个核细胞分离纯化主要的单核细胞<ol><li>用坎磁珠分离外周血单个核细胞从分离的单核细胞一个泛单核细胞分离试剂盒： <ol><li>通过30微米尼龙网（预分离器，30微米）通过细胞，以消除可能的细胞团块。 </li><li>使用血细胞计数器测定细胞数。 </li><li>离心细胞悬浮液在300×g下在室温（RT）下10分钟。吸上清完全。 </li><li>重悬细胞沉淀中加入30μl的PBS / EDTA / BSA缓冲液每10共7个细胞。 </li><li>加入10微升的FcR每10共7个细胞阻断剂。 </li><li>加入10μl生物素抗体鸡尾酒每10共7个细胞。 </li><li>拌匀，静置5分钟，在2-8°C。 </li><li>加入30微升缓冲液，每10 7细胞总数。 </li><li>加入20微升的每10 7总细胞抗生物素微珠。 </li><li>拌匀，静置10分钟，在2-8°C。 </li><li>悬浮高达10 8个细胞于500μl的PBS / EDTA / BSA缓冲液。 </li></ol></li><li> 磁组合模式振动性<ol><li>因此总细胞数，选择合适的MACS柱和MACS分离器（MACS见列数据表）。 </li><li>将列在MACS分离器的磁场。 </li><li>通过用PBS / EDTA / BSA缓冲液适量漂洗制备柱（MS色谱柱：500微升; LS柱：3ml）溶液。 注 ：列是“流停止”和不干涸。 </li><li>应用细胞悬液上柱。收集流过含有未标记细胞，占富含单核细胞部分。 </li><li>用PBS / EDTA / BSA缓冲液（500微升的MS和3毫升每洗LS）洗柱3倍。 </li><li>收集穿过，代表富集的单核细胞未标记细胞，并加入到流通的第3.2.4节。 注：通过添加PBS / EDTA / BSA缓冲液等份，只有当水库栏为空洗柱。 </li><li> （可选）从删除列分离器并将其放置在一个合适的收集管。吸取缓冲到柱（MS色谱柱：1毫升; LS列5毫升）。立即冲洗掉磁标记nonmonocyte细胞力推柱塞入列。 </li><li>培养物中纯化的完全RPMI-1640的单核细胞，用10％的人血清和抗生素。 </li></ol></li></ol> 4。从全血中分离纯化主要的单核细胞<ol><li>使用全血单核细胞分离试剂盒按照制造商的说明净化从全血中的单核细胞。 </li></ol> 5。纳米颗粒内在血液中白细胞和单核细胞纯化<ol><li>板5×10 5个细胞/ ml的分离的PBMC或纯化的CD14阳性单核细胞在12孔板（1毫升/孔）。 </li><li>涡流FITC-SiO 2的纳米颗粒储液，重悬在完全培养基中，以100nM的工作浓度。 </li><li>加入10微升的工作nanop的文章悬浮液（100纳米）对处理过的样品，达到1纳米的纳米颗粒的最终浓度。添加完全培养基为未处理的对照组相同体积到各孔中。 </li><li>后1小时内在收集的样品在1.5ml聚丙烯管中，在6000×g离心在RT离心他们3分钟。 </li><li>用1毫升在6000 XG在室温下运行缓冲液3分钟的洗涤沉淀。 </li><li>重悬沉淀在200μl缓冲液中。这些细胞是现在准备读通过流式细胞仪。 </li></ol> 6。原发性混合胶质细胞培养隔离（见Bertero 等 。7） 7。 Replating混合合流胶质细胞培养<ol><li>用10毫升的PBS洗一件T-75瓶一次（W / O型的Ca 2 + / Mg 2 +的），然后加入10维尔烯毫升和孵化在37℃5-7分钟。 </li><li>吸取上清上下数次，以从T-75的表面分离的细胞，溶解细胞聚集。 </li><li>转移在15毫升聚丙烯管中的细胞悬浮液。收集少量的细胞悬液计数细胞，血球。 </li><li>离心细胞悬浮液5分钟，在300×g离心于RT。 </li><li>去除上清，重悬沉淀以5×10 5细胞/胶质完全培养基毫升的最终浓度。 </li><li>制版为0.5ml /孔在24孔板中，并让细胞沉降纳米颗粒处理之前2-4小时。 </li></ol> 8。纳米颗粒内在的小胶质细胞<ol><li>加入500μl完整胶质培养基（对于未治疗的对照）或500微升的在完整胶质培养基2倍罗丹明的SiO 2纳米颗粒悬浮液（对于处理过的样品）到每个孔中。 </li><li>移液器的细胞，允许非快速附着的细胞在选定的时间点支队和收集他们在2毫升聚丙烯管。 </li><li>加入500μl维尔烯的每个孔中，培养板在37℃下5分钟，然后分离剩余的细胞部分和每个样品与上一步骤的相应nonadhering细胞收集。 </li><li>离心样品为3分钟，在300×g离心于RT。 </li><li>重悬沉淀在100μlAutoMACS运行缓冲液。 </li></ol> 9。与细胞CD11b-VioBlue染色<ol><li>加入10微升VioBlue-CD11b的人/鼠抗体100微升细胞悬液（一点11分比）的运行缓冲液孵育10分钟，在4°C。 </li><li>加入1 ml缓冲液和混合细胞悬液。 </li><li>离心每个样品为3分钟，在300×g下。 </li><li>弃上清，悬浮颗粒在100-150微升电泳缓冲液中。 </li><li>读出的样品的流式细胞仪（存储样品在4℃下的最后两个步骤之间）。 重要提示 ：分析样品前，进行重叠信号的赔偿我不同的荧光染料（荧光标记的纳米颗粒或抗体）n之间发射光谱观测。 </li></ol> 10。光谱溢出补偿<ol><li>打开“仪器设置”对话框（存在于所有的仪器软件程序）。 注意：如果使用从一个报道，在材料表不同的乐器，请参阅仪器说明书的具体的收购细节。 </li><li>在低温流体的速度（如果允许的仪器）收购未处理的样品（不含纳米颗粒）。在采集，通过调节电压通道手动调节前向和侧向散射（FSC和SSC，分别）。 注意：修改SSC和FSC轴刻度，以最好地显现完整的细胞群中的情节。一种仪器设置的模板为每个感兴趣的细胞类型可以被创建，保存并用于未来的收购。 </li><li>打开特定的“区域”选项卡中的软件。各地所需的人口画出一个合适的大区（“门”）。 注意：纳米颗粒的内化诱导该细胞的SSC移位。如果门已围绕感兴趣的细胞群过于紧密的限制，所获得的数据可能会错过要检测的细胞的一部分。 </li><li>用两个未染色样品，一是用作空白样品和一个用于对PI染色（可选）补偿，用适当的荧光染料进行补偿每个荧光通道单一染色样品。 注意：使用对于每个荧光标记的抗体在实验中标记要使用一个不同的1.5毫升样品管中。 </li><li>在“仪器设置”对话框中打开“补偿”选项卡。增加或收购时降低补偿系数，直到溢出通道的荧光强度大致相同的荧光POS可持续的竞争和人口负。 </li><li>获得纳米粒子处理过的样品后的补偿进行了调整。 </li></ol>

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作者声明美天旎生物技术有限公司赞助提交本手稿。 HiQ的纳公司（ <a href="http://www.hiq-nano.com/index.html">http://www.hiq-nano.com/index.html</a> ）是纳米技术分拆公司从IIT出现。

实验方案提出了要考虑的非常重要的点。这是非常重要的，在4℃（冰上），并可能在黑暗中，在所有的染色步骤，工作，因为较高的温度和灯可在染色产量产生负面影响。纳米粒子可以被超声，只是在使用前进行更好的悬浮。 正确的流式细胞仪分析需要一个正确的校准在不同的通道。每一个实验性会议之前应进行仪器的校准。除了用仪器的技术问题，可以有也问题与抗体标记。它是强制性的，以使用所述抗体在合适的浓度。如果浓度过高或过低，不满意的信号强度可以是结果。 该技术关注的缺点与单分散样品工作的需要，无法本地化网站上的信号（ 即不同的细胞区室）的起源。也有在荧光染料的选择一些限制在组合使用：激发和发射带的波长，必须充分分开，以允许其适当的测量。如果所使用的抗体谱重叠，一个正确的补偿是必需的。 流式细胞仪是一种功能强大的方法在存在或不存在的纳米颗粒的细胞分析。这种技术允许一个小区的多参数的研究中，高数量的事件检测，分析（多于1,000个细胞/秒），可重复性和统计读数的快速性。样品可以在不失去细胞活力进行处理。 通过使用荧光标记的纳米颗粒，可以限定和量化其内化于细胞亚群，这是由暴露在细胞膜上的特异性标志物鉴定。晶胞参数可以在的存在改变<html> pecific纳米粒子。取决于研究的目的，这些变化可以被用来识别特定的现象，如细胞比例与增加纳米颗粒的内化速率增加的侧散射。 细胞表面的纳米颗粒诱导的修饰也可以代表技术的限制。出于这个原因，细胞膜受体周转必须始终采取考虑和可能预先知道精确地表征感兴趣的细胞群。膜渗透的纳米颗粒重载样品中的极端损伤可导致细胞死亡。 纳米材料的剂量依赖性毒性应该为每个细胞群进行实证检验。明确定义的死细胞必须被排除在荧光定量。例如，膜联蛋白V / PI染色是其中的几种方法通常用来检测两个坏死和凋亡的细胞之一。 “&gt;表达GFP的原代细胞也是一个强大的工具来选择一个特定的细胞亚群，并收集数据，而无需预先标注。互补的荧光纳米颗粒的组合允许一个非常快速和精确的细胞纳米粒子相互作用的定量。药物或基因传递被认为是由纳米颗粒的应用能够释放一种特定药物负载到选择的组织在未来得到改善。 纳米粒子的制剂的特异性递送载体和/或免疫调节剂的就业需要的生物学环境的知识（ 即通过流式细胞术），来研究细胞的纳米粒子的相互作用。

纳米材料是当今鼓舞人心的生命科学家的兴趣的潜在应用到生物医学1。各种各样的有机和无机材料都可以用于制造纳米结构具有不同的形状，物理和化学特性。在这些结构中，球形的工程纳米颗粒表现出对诊断和转化医学2的巨大潜力。他们的核心和表面的设计是由一个可能的应用驱动，它意味着靶细胞反应的深入研究纳米以下的联系和相互作用。这被认为是故意给予人类受试者的纳米粒子会在与多种类型的免疫细胞直接接触。他们的责任，以保持身体的完整性，使他们在调查3纳米医学的一个重要课题。 先天免疫系统的细胞的部分主要是由吞噬表示核细胞。其中，单核细胞/巨噬细胞谱系的细胞，包括中枢神经系统常驻小胶质细胞，在免疫防御4,5中发挥关键作用。它们能够引发几小时内保护性反应与异物碰到后。此外，单核细胞中的坐标，并通过细胞因子的释放指示的适应性免疫反应。即使在工程材料，它是由免疫系统6在很大程度上被认为是“非自身”的存在下发生的所有这些事件。 其中历史上使用的免疫细胞分析方法，流式细胞仪代表了最有力的工具之一。此外，技术的特定的免疫细胞亚群（通常利用排除或一个单一的膜蛋白的存在下）的鉴定或纯化的可用性允许有一定的纳米颗粒在那个特定主ce的效果的精确调查LL型7。然而，细胞可暴露于纳米颗粒后呈现生理和形态的改变。以及，纳米颗粒可能会干扰特定的光学参数，例如光的吸收或发射在限定波长，影响了得到的结果8。所以，使用和经典的免疫学检测，以新材料的研究最终改编的限制应予以考虑。 本工作涉及原发性免疫细胞通过流式细胞术纳米粒子的相互作用的检测。为了解决这个问题，50纳米的FITC的SiO 2纳米粒子用作纳米材料模型来描述方法。二氧化硅颗粒可以在纳米尺度度量来制造在一个非常精确的方式。尺寸，形状和表面性质，如电荷或疏水性，可以精细地调谐，以增加其生物相容性9。二氧化硅的许多功能2纳米粒子使他们能够作为一个模型用于药物递送颗粒10。此外，荧光染料或量子点可以被截留或链接到这些颗粒提供有益的纳米工具，用于成像目的11。

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			<td style="width:107px;">Gibco</td>
			<td style="width:119px;">14170-088</td>
			<td style="width:256px;">Warm in 37 &#176;C water bath before use</td>
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			<td>Gibco</td>
			<td>A10491-01</td>
			<td>Warm in 37 &#176;C water bath before use</td>
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			<td height="21" style="height:21px;">DMEM, High Glucose, phenol red</td>
			<td>Gibco</td>
			<td>41966-029</td>
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			<td>Gibco</td>
			<td>15140-122</td>
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			<td>Gibco</td>
			<td>15710-049</td>
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			<td>16050-122</td>
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			<td>21985-023</td>
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			<td>15090-046</td>
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			<td>34005100</td>
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			<td>15040-033</td>
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			<td>D5025-150KU</td>
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			<td height="21" style="height:21px;">CD11b-VioBlue human &#38; mouse</td>
			<td style="width:107px;">Miltenyi Biotec</td>
			<td>130-097-336</td>
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			<td style="width:107px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-091-376</td>
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			<td height="21" style="height:21px;width:272px;">autoMACS Rinsing Solution&#160;</td>
			<td style="width:107px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-091-222</td>
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			<td height="21" style="height:21px;width:272px;">Running buffer</td>
			<td style="width:107px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-092-747</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:272px;">autoMACS Running Buffer</td>
			<td style="width:107px;">Miltenyi Biotec</td>
			<td>130-091-221</td>
			<td></td>
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			<td height="21" style="height:21px;width:272px;">Human Pan monocyte isolation kit&#160;</td>
			<td style="width:107px;">Miltenyi Biotec</td>
			<td>130-096-537</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Whole Blood Column Kit</td>
			<td style="width:107px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-093-545</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Whole Blood CD14 MicroBeads, human</td>
			<td style="width:107px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-090-879</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:272px;">MS Column</td>
			<td style="width:107px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-042-201</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:272px;">LS Column</td>
			<td style="width:107px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-042-401</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:272px;">MidiMACS Separator</td>
			<td style="width:107px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-042-302</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:272px;">MiniMACS Separator</td>
			<td style="width:107px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-042-102</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Red Blood Cell Lysis Solution&#160;</td>
			<td style="width:107px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-094-183</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Pre-Separation Filters 30 &#181;m</td>
			<td style="width:107px;">Miltenyi Biotec</td>
			<td>130-041-407</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:272px;">MACSQuant Analyzer flow cytometer</td>
			<td style="width:107px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-092-197</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:272px;">MACSQuant Calibration Beads</td>
			<td style="width:107px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-093-607</td>
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			<td height="21" style="height:21px;">Ficoll-Paque Premium</td>
			<td style="width:107px;">GE Healthcare</td>
			<td style="width:119px;">GEH17544202</td>
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			<td height="44" style="height:44px;width:272px;">FITC-SiO2 nanoparticles (50nm, +45mV)</td>
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			<td style="width:119px;"></td>
			<td></td>
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			<td height="46" style="height:46px;width:272px;">Rhodamine-SiO2 nanoparticles (50nm, +45mV)</td>
			<td style="width:107px;">HiQ-Nano Company</td>
			<td style="width:119px;"></td>
			<td></td>
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			<td height="42" style="height:42px;width:272px;">12-well plate Falcon</td>
			<td style="width:107px;">Becton Dickinson</td>
			<td style="width:119px;">353043</td>
			<td></td>
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detection of fluorescent nanoparticle interactions with primary immune cell subpopulations by flow cytometry

工程纳米颗粒被赋予了非常有前途的属性，治疗和诊断的目的。这项工作介绍了一种快速，可靠的分析方法，通过流式细胞仪研究纳米粒子的相互作用与免疫细胞。初级免疫细胞可以容易地从人或小鼠组织中由抗体介导的磁性隔离纯化。在第一个实例，在流式细胞仪上运行的不同的细胞群可以通过前向散射光（FSC），其正比于细胞大小和侧向散射光（SSC），与细胞内部的复杂性相关的区别开来。此外，对特定细胞表面受体的荧光标记的抗体允许几个亚群在同一样品中的鉴别。通常情况下，当细胞受到外界刺激会改变他们的生理和形态的状态提升所有这些功能而有所不同。在这里，50nm的FITC-SiO 2的纳米颗粒被用作模型，以确定第Ë内化纳米材料在人体血液中的免疫细胞。细胞荧光和侧向散射光增加孵育纳米颗粒后使我们能够确定时间和纳米颗粒 - 细胞相互作用的浓度依赖性。此外，这样的协议可以扩展到调查罗丹明的SiO 2纳米粒子的相互作用与初级小胶质细胞，中枢神经系统中驻留的免疫细胞，分离出特异性表达的绿色荧光蛋白（GFP）在单核细胞/巨噬细胞系的突变小鼠。最后，流式细胞术相关的纳米颗粒的内化到细胞中的数据已经证实了通过共聚焦显微镜。

流式细胞术是一种有用的工具，以鉴定和表征不同的细胞，它是所选择的技术，以识别特定的免疫细胞，如单核细胞，粒细胞，T细胞，B细胞，自然杀伤（NK）细胞，树突状细胞（DC），和白细胞等亚群。 在为了更好地响应于纳米粒子表征的白血细胞的行为，我们进行内在化分析原发性白细胞从健康供体的血液（ 图1和2），并与人单核细胞系中分离（THP-1细胞， 图3） 。 据报道在图1A，三大血白细胞亚群外周血单个核细胞后分离明确确定由前向和侧向散射。而且，之后的FITC-SiO 2的处理，淋巴细胞（灰色），单核细胞（蓝色）和粒细胞（红色）有一个不同的n通过绿色荧光强度（ 图1B）所示anoparticle内化率。所描述的协议允许从PBMC中初级CD14阳性单核细胞的纯化。 图2A报告流式细胞仪的CD14 +单核细胞中FITC-SiO 2的存在下的点图。 图2B显示了FITC-SiO 2的内化量化在相同的细胞和表达对数标度直方图。 类似的内化实验在用FITC-SiO 2的纳米颗粒浓度的增加处理的THP-1单核细胞进行的。未经处理的细胞作为阴性对照。 图3A示出了剂量依赖性增加在侧散射与未改变的前进中的THP-1细胞系的散射。在图3B中 ，连同SSC和FSC在每个FITC-SiO 2的纳米颗粒浓度测试的直方图，平均荧光强度（MFI）的量化呈现。此数据表明，用FITC-SiO 2的纳米颗粒治疗诱导剂量依赖性的内在化的单核细胞通过细胞内的粒度的提高（侧面散射）和荧光（绿色通道）高亮显示。 以获得进一步的见解免疫细胞和纳米颗粒之间的相互作用的类型，原发性混合胶质细胞培养物中分离和小胶质细胞，中枢神经系统中驻留的免疫细胞，进行纯化。利用表达绿色荧光的小胶质细胞的转基因小鼠模型允许不同的神经炎症机制的可视化。该转基因小鼠B6.129P-CX3CR1 tm1Litt /法官在这项工作中使用的表达绿色荧光蛋白（GFP）CX3CR1子12的控制下。 7天之后在体外 （DIV），荧光显微镜显示了一个混合初级神经胶质培养具有大量星形胶质细胞（GFP阴性细胞粘附次）和一些绿色细胞（GFP阳性， 图5A）。 GFP - - （星形胶质细胞和其他神经胶质细胞），第二不同组的小胶质细胞CD11b + GFP +细胞中，并且所述第一CD11b的：在此小鼠模型中，3胶质亚群可以通过流量与单一的CD11b抗体染色区分术第三个的CD11b + GFP -亚群（ 图4A）。后两个亚群都能够通过将GFP +人口（占巡逻不成熟的小胶质细胞通过CX3CR1启动子的转录）内化纳米粒子有轻微的效率更高，如通过流式细胞仪分析（ 图4B）。在发生内化，可以进一步通过共聚焦显微镜使用罗丹明的SiO 2纳米粒子的相同最终浓度为如图5B所示核实。 图1。在孤立的血液白细胞A）代表前向散射（FSC） 与侧散射FITC-SiO 2的纳米颗粒内在 （SSC），流式细胞仪的菲可-帕克分离血液中的白细胞点图B）绿色荧光叠加直方图三大血白细胞的阴谋细胞亚群在1nm FITC-SiO 2的纳米颗粒（45毫伏），1小时出现。 <a href="https://www.jove.com/files/ftp_upload/51345/51345fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" fo:content-width="5in" fo:src="/files/ftp_upload/51345/51345fig2highres.jpg" src="/files/ftp_upload/51345/51345fig2.jpg" /> <strong&gt;的图2。在CD14 FITC-SiO 2的纳米颗粒内在+单核细胞纯化A）代表前向散射（FSC） 与侧散射（SSC），流式细胞术纯化的CD14阳性单核细胞的点图B）绿色荧光直方图中存在的纯化单核细胞亚群的阴谋1nm的FITC-SiO 2的纳米颗粒（45毫伏）1小时。 <a href="https://www.jove.com/files/ftp_upload/51345/51345fig2highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" fo:content-width="5in" fo:src="/files/ftp_upload/51345/51345fig3highres.jpg" src="/files/ftp_upload/51345/51345fig3.jpg" /> 图3。对THP-1细胞A）代表前向散射（FSC） 对侧面散射问题的FITC-SiO 2的纳米颗粒的内化作用克（SSC）的流式细胞术的THP-1单核细胞系中的点图，以下FITC-SiO 2的纳米颗粒浓度的增加B）的浓度依赖性的侧散射的变化（SSC）的1小时暴露，前向散射（FSC）和绿荧光FITC-SiO 2的纳米颗粒（45毫伏）1小时的存在。 <a href="https://www.jove.com/files/ftp_upload/51345/51345fig3highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" fo:content-width="5in" fo:src="/files/ftp_upload/51345/51345fig4highres.jpg" src="/files/ftp_upload/51345/51345fig4.jpg" /> 图4。罗丹明的SiO 2纳米颗粒内在分为原发性小胶质细胞从B6.129P-CX3CR1 tm1Litt / J小鼠中分离A）绿色荧光蛋白（GFP） 与细胞CD11b-VioBlue流式细胞公关的点图imary混合胶质细胞从B6.129P-CX3CR1 tm1Litt / J小鼠中分离。细胞CD11b + GFP +和CD11b + GFP -人口正在30中所示的右上和右下象限，分别为B）在1nm罗丹明的SiO 2纳米颗粒（45毫伏）存在小胶质细胞亚群的红色荧光叠加直方图。分钟（红色柱状图） 与控制（灰度直方图）。 <a href="https://www.jove.com/files/ftp_upload/51345/51345fig4highres.jpg" target="_blank">请点击此处查看此图的放大版本。</a> <img alt="图5" fo:content-width="5in" fo:src="/files/ftp_upload/51345/51345fig5highres.jpg" src="/files/ftp_upload/51345/51345fig5.jpg" /> 图5。可视化的GFP +小胶质细胞（A）荧光显微镜在7 DIV和（B）共聚焦显微镜罗丹明-SiO 2的纳米颗粒内在（红色箭头）的GFP +小胶质细胞。 <a href="https://www.jove.com/files/ftp_upload/51345/51345fig5highres.jpg" target="_blank">请点击此处查看此图的放大版本。</a>

Watch this Scientific Journal Video about Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry at JoVE.com

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

分析与免疫细胞的流式细胞仪检测亚组人群纳米粒子的相互作用。

检测荧光纳米粒子的相互作用与原发性免疫细胞亚群流式细胞仪的

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

detection-fluorescent-nanoparticle-interactions-with-primary-immune

Research

JoVE Journal

Immunology and Infection

17.4K 次观看.  Istituto Italiano di Tecnologia. 分析与免疫细胞的流式细胞仪检测亚组人群纳米粒子的相互作用。 

视频: Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

在固定和活细胞，流式细胞仪测量钙蛋白酶活性

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

科研

免疫与感染

Quantitative Assessment of Immune Cells in the Injured Spinal Cord Tissue by Flow Cytometry:  a Novel Use for a Cell Purification Method

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true quantitative analysis of cellular inflammation. Flow cytometry is a quick alternative method to quantify immune cells in the injured brain or spinal cord tissue. Historically, flow cytometry has been used to quantify immune cells collected from blood or dissociated spleen or thymus, and only a few studies have attempted to quantify immune cells in the injured spinal cord by flow cytometry using fresh dissociated cord tissue. However, the dissociated spinal cord tissue is concentrated with myelin debris that can be mistaken for cells and reduce cell count reliability obtained by the flow cytometer. We have advanced a cell preparation method using the OptiPrep gradient system to effectively separate lipid/myelin debris from cells, providing sensitive and reliable quantifications of cellular inflammation in the injured spinal cord by flow cytometry. As described in our recent study (Beck &amp; Nguyen et al., Brain. 2010 Feb; 133 (Pt 2): 433-47), the OptiPrep cell preparation had increased sensitivity to detect cellular inflammation in the injured spinal cord, with counts of specific cell types correlating with injury severity. Critically, novel usage of this method provided the first characterization of acute and chronic cellular inflammation after SCI to include a complete time course for polymorphonuclear leukocytes (PMNs, neutrophils), macrophages/microglia, and T-cells over a period ranging from 2 hours to 180 days post-injury (dpi), identifying a surprising novel second phase of cellular inflammation. Thorough characterization of cellular inflammation using this method may provide a better understanding of neuroinflammation in the injured CNS, and reveal an important multiphasic component of neuroinflammation that may be critical for the design and implementation of rational therapeutic treatment strategies, including both cell-based and pharmacological interventions for SCI.

Quantification of cellular inflammation in the injured/pathological CNS by flow cytometry is complicated by lipid/myelin debris that can have similar size and granulation to cells, decreasing sensitivity/accuracy. We have advanced a cell preparation method to remove myelin debris and improve cell detection by flow cytometry in the injured spinal cord.

免疫细胞在受伤的脊髓组织流式细胞仪定量评估：一个细胞的分离纯化方法的新颖使用

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true ...

Non-surgical Intratracheal Instillation of Mice with Analysis of Lungs and Lung Draining Lymph Nodes by Flow Cytometry

Phagocytic cells such as alveolar macrophages and lung dendritic cells (LDCs) continuously sample antigens from the alveolar spaces in the 
lungs. LDCs, in particular, are known to migrate to the lung draining lymph nodes (LDLNs) where they present inhaled antigens to T cells initiating an 
appropriate immune response to a variety of immunogens1,2. To model interactions between the lungs and airborne antigens in mice, antigens can be 
administered intranasally1,3,4, intratracheally5 or as aerosols6. Delivery by each route involves distinct technical skills and limitations that need to be 
considered before designing an experiment. For example, intranasal and aerosolized exposure delivers antigens to both the lungs and the upper respiratory 
tract. Hence antigens can access the nasal associated lymphoid tissue (NALT)7, potentially complicating interpretation of the results. In addition, swallowing, 
sneezing and the breathing rate of the mouse may also lead to inconsistencies in the doses delivered. Although the involvement of the upper respiratory tract may 
be preferred for some studies, it can complicate experiments focusing on events specifically initiated in the lungs. In this setting, the intratracheal (i.t) 
route is preferable as it delivers test materials directly into the lungs and bypasses the NALT. Many i.t injection protocols involve either blind intubation of the 
trachea through the oral cavity or surgical exposure of the trachea to access the lungs. Herein, we describe a simple, consistent, non-surgical method for i.t 
instillation. The opening of the trachea is visualized using a laryngoscope and a bent gavage needle is then inserted directly into the trachea to deliver the 
innoculum. We also describe procedures for harvesting and processing of LDLNs and lungs for analysis of antigen trafficking by flow cytometry.

We illustrate non-surgical delivery of test materials into the lungs of anesthetized mice via the trachea. This method permits lung exposure to bacterial and viral pathogens, cytokines, antibodies, beads, chemicals, or dyes. We further describe harvesting and processing of lungs and lung draining lymph nodes (LDLNs) for flow cytometry.

非手术治疗的小鼠气管内滴入肺和肺淋巴结流式细胞仪分析

Phagocytic cells such as alveolar macrophages and lung dendritic cells (LDCs) continuously sample antigens from the alveolar spaces in the 
lungs. LDCs, ...

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

流式细胞仪分析小鼠主动脉内流的免疫细胞

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic &beta;-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

人类<em在体外承认免疫失衡的早期国家禁止作为筛检工具

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of phenol soluble modulins (PSMs) and other toxins produced and secreted by Staphylococcus aureus on neutrophils. To study the effects of the PSMs on neutrophils we isolate fresh neutrophils using density gradient centrifugation. These neutrophils are loaded with a dye that fluoresces upon calcium mobilization. The activation of neutrophils by PSMs initiates a rapid and transient increase in the free intracellular calcium concentration. In a flow cytometry experiment this rapid mobilization can be measured by monitoring the fluorescence of a pre-loaded dye that reacts to the increased concentration of free Ca2+. Using this method we can determine the PSM concentration necessary to activate the neutrophil, and measure the effects of specific and general inhibitors of the neutrophil activation.	
	To investigate the expression of the PSMs in the intracellular space, we have constructed reporter fusions of the promoter of the PSM&alpha; operon to GFP. When these reporter strains of S. aureus are phagocytosed by neutrophils, the induction of expression can be observed using fluorescence microscopy.

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of PSMs and other toxins secreted by Staphylococcus aureus on neutrophils using flow cytometry and fluorescence microscopy.

相互作用的研究<em&gt;金黄色葡萄球菌</em与中性粒细胞通过流式细胞仪和时间推移显微镜

We present methods to study the effect of phenol soluble modulins (PSMs)  and other toxins produced and secreted by Staphylococcus  aureus on neutrophils. ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

荧光激活细胞分选从小鼠骨髓浆树突状细胞的分离纯化

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) cycles between a tsetse vector and a mammalian host. It evades the mammalian host immune system by periodically switching the dense, variant surface glycoprotein (VSG) that covers its surface. The detection of antigenic variation in Trypanosoma brucei can be both cumbersome and labor intensive. Here, we present a method for quantifying the number of parasites that have 'switched' to express a new VSG in a given population. The parasites are first stained with an antibody against the starting VSG, and then stained with a secondary antibody attached to a magnetic bead. Parasites expressing the starting VSG are then separated from the rest of the population by running the parasites over a column attached to a magnet. Parasites expressing the dominant, starting VSG are retained on the column, while the flow-through contains parasites that express a new VSG as well as some contaminants expressing the starting VSG. This flow-through population is stained again with a fluorescently labeled antibody against the starting VSG to label contaminants, and propidium iodide (PI), which labels dead cells. A known number of absolute counting beads that are visible by flow cytometry are added to the flow-through population. The ratio of beads to number of cells collected can then be used to extrapolate the number of cells in the entire sample. Flow cytometry is used to quantify the population of switchers by counting the number of PI negative cells that do not stain positively for the starting, dominant VSG. The proportion of switchers in the population can then be calculated using the flow cytometry data.

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

African trypanosomes grown in vitro undergo antigenic variation at a low rate, such that populations are made up of parasites expressing a dominant variant surface glycoprotein (VSG) type and a small population of "switched" variants. This protocol describes a fast method for detecting and quantifying these populations.

检测<em&gt;布氏锥虫</em&gt;变体表面糖蛋白通过磁性细胞分选和流式细胞仪开关

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

小鼠与人肺泡巨噬细胞的分离和体外培养

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Discrimination of Seven Immune Cell Subsets by Two-fluorochrome Flow Cytometry

Immune cell characterization heavily relies on multicolor flow cytometry to identify subpopulations based on differential expression of surface markers. Setup of a classic multicolor panel requires high-end instruments, custom labeled antibodies, and careful study design to minimize spectral overlap. We developed a multiparametric analysis to identify major human immune populations (CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, NK cells and monocytes) in peripheral blood by combining seven lineage markers using only two fluorochromes. Our strategy is based on the observation that lineage markers are constantly expressed in a unique combination by each cell population. Combining this information with a careful titration of the antibodies allows investigators to record five additional markers, expanding the optical limit of most flow cytometers. Head-to-head comparison demonstrated that the vast majority of immune cell populations in peripheral blood can be characterized with comparable accuracy between our method and the classic &#34;one fluorochrome-one marker approach&#34;, although the latter is still more precise for identifying populations such as NKT cells and &#947;&#948; T cells. Combining seven markers using two fluorochromes allows for the analysis of complex immune cell populations and clinical samples on affordable 6-10 fluorochrome flow cytometers, and even on 2-3 fluorochrome field instruments in areas with limited resources. High-end instruments can also benefit from this approach by using extra fluorochromes to accomplish deeper flow cytometry analysis. This approach is also very well suited for screening several cell populations in the case of clinical samples with limited number of cells.

Here, we present a flow cytometric protocol to identify CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, NK cells and monocytes in human peripheral blood by using only two fluorochromes instead of seven. With this approach, five additional markers can be recorded on most flow cytometers.

双氟铬流式细胞仪对七种免疫细胞子集的判别

Immune cell characterization heavily relies on multicolor flow cytometry to identify subpopulations based on differential expression of surface markers. ...