这个项目是由卫生部（5 P20 RR016461和8 P20 GM103499）的国家机构的资金支持，美国国家科学基金会（EPS-0903795）。通过弗曼大学的研究和专业成长和弗曼优势奖项提供了进一步的支持。

这项研究进行了严格按照指南中的美国国立卫生研究院的实验动物的护理和使用的建议。动物方案得到批准弗曼大学机构动物护理和使用委员会（许可证号:A3242-01）。所有材料在这个协议中使用的设备能材料和设备的表中找到，而使用的缓冲器可缓冲和解决方案的表中找到。 1.单细胞悬液的淋巴细胞从小鼠脾脏<ol><li>通过二氧化碳窒息牺牲老鼠。确认安乐死颈椎脱位。 注意:使用脾脏从同一性别两个6-8周龄同基因小鼠（例如，C57B / 6）。通常情况下，二脾脏被处理。 </li><li>喷动物大汗，用70％的乙醇溶液和定向，使得头朝左。 </li><li>使用镊子，提起动物的皮肤向上和远离吨他的身体。使用剪刀，通过附近的动物的腹部皮肤切开一个小切口。用手指，拉出槽口的每一侧，以从颈部腹膜暴露于后​​腿的开头。脾应可见直接腹膜下方。 </li><li>使用镊子，提起腹膜，并做一个小切口，显露脾。用钳子取出脾并用剪刀戏弄离开任何脂肪和结缔组织。 </li><li>将脾脏到50毫升锥形管中含有10毫升的PBS补充有2％胎牛血清（以下简称为PBS + 2％）。保持管在冰上直至准备开始单细胞悬浮液。 注意:典型的恢复是每脾之间60-90百万个细胞，并且通常，需要2百万细胞，每个样品。执行所有以下步骤在组织培养罩。 </li><li>滗脾脏进入无菌100mm组织培养皿。 </li><li>获得两个磨砂显微镜载玻片和保持牛逼他滑动，使得两个粗糙磨砂表面面对向内朝向彼此。湿润PBS + 2％的溶液中的幻灯片倒入培养皿。 </li><li>保持幻灯片的磨砂表面之间的脾，与仍然淹没的边缘，并通过移动来回滑动相互对抗（ 图1A）轻轻研磨脾脏。这些细胞会落入培养皿。继续打磨，直到所有的细胞已被释放，脾的遗骸出现白色（ 图1B）。 </li><li>绘制出的细胞悬浮液在吸管，并通过70微米的过滤器过滤它慢慢倒入一个新的无菌50ml锥形管中。需要注意的是小件红髓将存在于过滤器（图2A）。 <ol><li>如果过滤器上升，轻轻将其提起略微以允许流体排出，通过与将过滤器放回50ml锥形管中并继续，根据需要重复这个过程。如果在处理超过5 spleENS，可能有必要分成两批，并使用一个新的过滤器的每个。 </li></ol></li><li>用3毫升注射器的止动部轻轻按压穿过滤网（ 图2B）含有剩余细胞红髓。一定要按下两个过滤器底部和侧面，以确保所有的红髓已通过过滤器（图2C）通过。 </li><li>添加10 mlof PBS + 2％的组织培养皿中，并用它来洗幻灯片，注射器限位装置，菜以回收剩余的细胞。通过相同的70微米的细胞过滤到50毫升锥形管传递此。这个步骤需要重复。 </li><li>离心过滤的细胞悬浮液在300×g离心5分钟，在4℃。 </li><li>轻轻倒出并弃去上清液，注意不要打扰颗粒。的PBS + 2％一丝金额将留在管。帽管和轻弹沉淀，直到沉淀完全重悬。 </li><li>裂解红细胞按广告丁2毫升的ACK裂解缓冲液（0.15M的氯化铵 ，1mM的KHCO 3，0.1mM的EDTA，pH为7.2，保存于4℃）每脾到锥形管并旋转和反转管，以确保所有的细胞接触到该ACK缓冲器接触。轻轻旋转管在室温1分钟。 </li><li>通过加入PBS + 2％管以中和该ACK缓冲器使细胞悬浮液的体积至50ml。倒置该管的10倍，离心5分钟，在300×g离心在4℃下。离心完成后，将沉淀应该是白色的（图3）。 </li><li>轻轻倒出并弃去上清液，注意不要打扰颗粒。离开的PBS + 2％的微量在管，帽管，并轻弹颗粒重悬了。 </li><li>加入10 mL的T细胞培养基[10％FBS的10mM HEPES（pH 7.0）中，2毫含有GlutaMAX，1mM丙酮酸钠，1×非必需氨基酸，1×青霉素/链霉素和50mMβ巯基乙醇〕，和进一步的轻轻悬浮颗粒上下吹打。然后通过一个新的70微米的过滤器过滤该悬浮液到一个新的50ml锥形管中。 </li><li>使用另一个5-10毫升的T细胞培养基冲洗原管，并通过70微米的过滤器通过该入含有过滤细胞的其余部分的管。如果在处理两个以上的脾脏，T细胞培养基的体积可增加到最大限度回收。 </li><li>保持细胞在冰上，直到准备染色。细胞应该算和可行性进行访问。 <ol><li>计数细胞，结合3 ml的细胞用24毫升的PBS + 2％和3 ml的0.4％台盼蓝。负载10毫升到这一种改进的Neubauer血细胞计数器的每个腔室。可行性的评估，台盼蓝染色，通常为85-95％。 </li></ol></li></ol> 2.活力和表面染色<ol><li>沉淀细胞，在300×g离心离心10分钟，在4℃。重悬的细胞在T细胞培养基至1×10 7个细胞/ ml的浓度。 TRANSF器2百万细胞（100毫升）中成U形底96孔组织培养板的孔中。 </li><li>离心96孔板10分钟，300×g离心在4℃下。 </li><li>井由孔板进水槽内轻弹除去液体从每个上清液（细胞沉淀将保留在孔）并在干净的纸巾涂抹在板。通过再悬浮他们在200μl的PBS洗涤细胞，然后离心以300×g离心在4℃下10分钟。除非另有说明执行所有洗涤这种方式。 注:请不要使用PBS + 2％，因为它会与定像PI染色干扰。 </li><li>通过轻弹板除去上清液，加入100毫升新制备的商用染色剂（ 例如 ，可固定红死细胞染色）的各孔。 <ol><li>通过稀释在DMSO活性染料原液1点10分，随后用1:10稀释在PBS使污渍。孵育板在4℃下避光30分钟。 </li></ol></li><li>离心在p迟在300×g离心10分钟，在4℃。在这一点上前进，执行与该板与组织文化引擎盖光了所有的工作。 </li><li>通过轻弹平板去除上清，再加入100毫升的Fc块解决方案。制作的Fc封闭液稀释的Fc块原液1:在PBS 100。 </li><li>要使用的稀释抗体表面染色（例如，抗CD4或抗CD8）在PBS中，加入100 ml的每个孔中。孵育96孔板30分钟，在4℃下避光。 注意:表面染色的抗体应滴定以获得最佳性能。通常使用1:200和1:400稀释，根据制造商的协议。 </li></ol> 3.细胞内染色的组蛋白H3 <ol><li>以下表面染色温育后，在PBS中洗细胞两次。 </li><li>最后一次洗涤后，加入100μl的4％多聚甲醛的每个孔中。孵育96孔板5分钟，在RT。使4％多聚甲醛通过dilut荷兰国际集团在PBS 16％多聚甲醛。使这个新鲜的。 </li><li>在PBS洗涤细胞两次。 </li><li>使彼尔姆/砌块溶液（股票彼尔姆溶液是PBS + 2％+ 0.02％的Triton X-100，保存于4℃）用正常兔血清的每100毫升的库存彼尔姆溶液将2ml。做足够的60毫升/样本。 </li><li>添加40毫升永久/块到每个样品孔拌匀轻轻吹打向上和向下，注意不要让泡沫。孵育板在室温在黑暗中45分钟。保存剩余的烫发/块步3.6。 </li><li>稀荧光缀合的组蛋白H3K4me1抗体在步骤3.5保留彼尔姆/砌块溶液，并添加10微升该稀释的每个孔中。轻轻吹打向上和向下混合。孵育96孔板在黑暗中进行1小时，在4℃。 注意:使用的H3K4me1抗体使用R-藻红蛋白缀合试剂盒按照制造商的说明，缀合。 </li><li>两次在PBS + 2％洗细胞。 </li><li>悬浮细胞在200μLPBS + 2％和转移样本，以FACS管用于流式细胞术分析。样品可以储存在4℃下在黑暗中。为了达到最佳效果分析样品在两天之内。单独使用染色的样品可以用作流式细胞补偿控制。 </li></ol>

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作者宣称，他们有没有竞争的财务权益。

我们开发了一个协议，允许染色质凝聚在T细胞的评估。它依赖于组蛋白H3抗体无法访问其表位易于在幼稚细胞的简单的观察，但在T细胞活化，这些相同的抗体能够结合其表位。通过比较MFI组蛋白H3的治疗组之间的染色，缩合或解聚的相对程度可被确定。我们已经使用该协议，以确定在胸腺细胞发育，并在T细胞活化4相对的结露状态。我们也使用该协议来研究用于控制解聚过程5所述的机制。 这个协议依赖于使用一个组蛋白H3抗体来检测染色质凝聚状态。由于是在T细胞活化4组蛋白修饰没有全球变化，我们能够使用H3K4me1抗体来评估染色质在本协议中的地位。我们已经使用了针对未修饰组蛋白H3抗体;然而，所产生的信号是弱得多的整体。根据我们的经验，提出对修饰的组蛋白H3抗体免疫工作更好，流式细胞仪检测，而抗未修改的组蛋白H3在Western印迹更好地工作。必须指出的是，也可以使用了针对其他组蛋白的抗体，尽管我们并没有试图它。 彼尔姆/砌块和组蛋白H3抗体步骤是在协议中最重要的步骤。需要使用的彼尔姆/砌块溶液量的库存彼尔姆溶液制成每次进行优化。这可以通过比较原液不同稀释度的性能来完成。一个典型的实验涉及幼稚T细胞的那些3小时（图4）激活的比较染色质状态。每个人都应该选择在产生变化最大的小额信贷机构的稀释的时间段进行分析。原液可以在PBS + 2％稀释由第一重悬细胞中高达100毫升的PBS + 2％步骤3.4之后，然后加入彼尔姆/砌块在步骤3.5以降低彼尔姆/砌块强度。类似的预试验应该使用新的批次的抗体被标记每次测试荧光缀合的组蛋白H3抗体的不同稀释。这些步骤是成功侦破凝结差异在T细胞活化早期尤为重要（ 例如，在3个小时的激活）。偶尔也有细胞没有得到透化，因而不会与组蛋白H3抗体染色。这可能是由于细胞没有悬浮以及加入烫发/块时，或者发生，如果烫发/块还不够强大。这些细胞会显示为可视化的组蛋白H3染色直方图时压在轴事件。由于这些事件将歪斜的整体MFI，这些事件可以从分析由gatin省略克组蛋白H3阳性细胞的正常分配。其他帮助可在故障排除指南（ 表1）中找到。 列入一个可固定死细胞染色允许对细胞活力在测定中评估。操作T细胞的活化，因为某些刺激可以诱导细胞死亡时，这绝对是至关重要的。在这样的情况下，该组蛋白H3抗体可以结合于死亡细胞组蛋白不同于活细胞，导致的结果的曲解。 这种协议被设计为在96孔板形式使用，允许的染色质状态的高通量分析。从6-8周龄雌性小鼠脾脏通常会产生使用的协议之间的60-90万个细胞。由于染色协议需要每个样品2百万细胞，人们可以很容易测定在单个96孔板中一式三份单个脾多个治疗组和时间点。有可能为perform每个样品更少细胞的协议;然而，由于离心步骤的数目和细胞的每个步骤的固有损耗，这是不可取的由多，以降低细胞的数目。我们已经成功地完成了每样品1百万个细胞的协议。 我们利用这个协议来检查CD4 + T辅助细胞和CD8 +细胞毒性T细胞染色质状态。这是可能的，因为该协议包括标准表面染色。该协议可以很容易地适合于染色质在其他淋巴细胞亚群通过使用抗人群特异性表面标志物的检查。该协议也很容易，只要适用于其他类型的细胞的抗体识别相关的表面标志是可用的，适当的固定/通透条件是已知的。

流式细胞术是用于在细胞群的多个物理和荧光参数的分析开发出一种基于激光的技术。这种技术可以作为悬浮细胞中的流体相遇流的激光器，令人兴奋的荧光标记上或在细胞内。这些标记然后发出被检测到并且通过光电倍增管定量的光。虽然流式细胞历来用于鉴定细胞群，它已被证明研究细胞的性能，包括细胞膜完整性，蛋白质-蛋白质相互作用和蛋白质运输1-3的阵列时是一种有用的技术。我们已经开发出一种协议，它允许这种技术也可以用于检测染色质在T细胞的状态，因为它们是在体外 4激活。我们也使用该协议来研究T细胞5的活化诱导染色质解聚的机制。 T细胞活化法制n和扩散是一个适当的免疫反应的关键。 T细胞，免疫系统内淋巴细胞的特定子集，需要进行适当的免疫应答和免疫记忆的发展。当抗原呈现在主要兼容性复杂的T细胞受体（TCR）位于静止的T细胞的细胞外表面（6中综述）的上下文激活被启动。这触发了一系列的T细胞内为最终结果的增加细胞内钙离子浓度7和所需的活化（以8-10中综述）转录因子的核转位的动态和高度有序分子事件。一旦被激活，T细胞获得对以白细胞介素-2（IL-2），利用所述JAK（JAK激酶）/ STAT一个有效的生长因子作出反应的能力（信号转导及转录激活因子的）途径，以驱动激活的T克隆增殖细胞11。简言之，将IL-2的刺激结果在STAT蛋白，一个家庭的潜胞质转录因子的磷酸化。一旦磷酸化，STAT蛋白二聚化，转位到细胞核并驱动的基因，包括那些涉及细胞周期进程的表达。在T细胞中，经由STAT5，这是需要的T细胞增殖12,13的IL-2的信号。 为了实现活化的T细胞的克隆扩增，那些没有经历抗原的TCR接合（幼稚T细胞）细胞，必须有一种机制来忽略的IL-2的强效作用。这是通过染色质状态的调节来实现的。幼稚T细胞具有稠染色质，禁止STAT5-DNA的接合应答IL-2刺激。在激活时，染色质decondenses和STAT5的可以访问的靶基因的启动子，允许克隆增殖4。有趣的是，这种变化在染色质状态是不依赖于组蛋白protei的表观遗传修饰NS（综述见14），我们观察到在T细胞活化4组蛋白修饰没有全球变化。 在执行这些研究中，我们发现，提出对组蛋白抗体也很难进入在幼稚细胞表位的，但在活化时能更容易地结合它们的表位4。因此，抗体结合到组蛋白用作读出为染色质凝聚状态。这里，我们提出的方法使用流式细胞术来检测荧光缀合组蛋白H3的抗体，以在T细胞评估染色质状态。在细胞群体染色质缩合测量为组蛋白H3染色的平均荧光强度（MFI）。在T细胞活化，对组蛋白H3染色的增加的MFI，表示染色质的解聚的上下文。除了通过细胞内的组蛋白H3的染色质检测情况，该协议还incorporaTES表面染色和可修复染色的活细胞，允许细胞亚群的分析。

<table border="0" cellpadding="0" cellspacing="0" style="width:584px;" width="585">
	<tbody>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">100mm tissue culture dish</td>
			<td style="width:83px;">BD Biosciences</td>
			<td style="width:119px;">353003</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Precleaned frosted microscope slides</td>
			<td style="width:83px;">Fisher Scientific</td>
			<td>12-550-343</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Sterile cell strainer, 70mm nylon mesh</td>
			<td style="width:83px;">Fisher Scientific</td>
			<td>22363548</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">3mL syringe, Luer-Lok tip</td>
			<td style="width:83px;">BD</td>
			<td>309585</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Falcon 50mL polypropylene conical tube</td>
			<td style="width:83px;">Corning</td>
			<td>352098</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">LIVE/DEAD fixable red dead cell stain kit</td>
			<td style="width:83px;">Life Technologies</td>
			<td>L23102</td>
			<td style="width:167px;">Life Technologies sells versions of this kit with different colors</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Fc Block</td>
			<td style="width:83px;">BD Biosciences</td>
			<td>553142</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">16% paraformaldehyde</td>
			<td style="width:83px;">Electron Microscopy Sciences</td>
			<td>15710</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Triton X-100</td>
			<td style="width:83px;">Fisher Scientific</td>
			<td>BP151-100</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Normal rabbit serum</td>
			<td style="width:83px;">Sigma-Aldrich</td>
			<td style="width:119px;">R9133</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Anti-H3K4me1 antibody</td>
			<td style="width:83px;">Abcam</td>
			<td>ab8895</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">R-Phycoerythrin conjugation kit</td>
			<td style="width:83px;">Abcam</td>
			<td>ab102919</td>
			<td style="width:167px;">Alternatively, the LYNX rapid RPE kit from AbD Serotec can be used</td>
		</tr>
	</tbody>
</table>

the use of flow cytometry to assess the state of chromatin in t cells

在一个适当的免疫应答，静止T细胞成为经抗原呈递到其抗原特异性T细胞受体激活。这导致只有那些T细胞而承受的受体识别抗原的克隆增殖。染色质解聚是T细胞活化的标志并且对于T细胞获得抗原接合之后增殖的能力是必需的。可以使用针对组蛋白抗体来检测这种变化中染色质凝聚。这些抗体不能绑定到他们的幼稚T细胞表位，以及他们可以在活化的T细胞。我们描述了如何同时染色T细胞特异性表面标记，跟踪生存能力有可以解决的死细胞染色，并通过组蛋白H3蛋白的细胞内染色质测量状态。染色的细胞通过流式细胞仪分析和染色质凝聚状态测量为组蛋白H3染色的平均荧光强度（MFI）。 Chromat在过程中的T细胞激活解聚是表现为增加的MFI

从一个C57B / 6小鼠的淋巴细胞根据该协议处理成单细胞悬浮液，并用标准血细胞计数器计数。细胞接种在一式三份以2×10在T细胞培养基6 / ml的在15ml锥形管中，并加以治疗，或在刺激1毫克/毫升的可溶性抗CD3抗体（克隆4C11）3小时，在37℃一个标准的组织培养孵化器。死细胞染色，然后细胞然后进行表面使用的FITC的CD8和APC CD4抗体按照协议染色。组蛋白辅助经流式细胞术（ 图4）进行分析。在这两种幼稚CD4 +和CD8 + T细胞的平均荧光强度（MFI）为低，表示稠染色质状态。作为细胞用抗CD3抗体激活MFI的增加显著（P &lt;0.001，学生t检验），表明已染色质解聚。 <img alt="图1" src="/files/ftp_upload/53533/53533fig1.jpg" /> 图1. 技术用于处理脾成使用磨砂显微镜载玻片单细胞悬浮液。（A）中的脾被压靠在两个显微镜载玻片的磨砂表面。 （ 二）幻灯片来回移动对对方释放淋巴细胞到100毫米的培养皿，直到脾脏的遗体是白色的。 <a href="https://www.jove.com/files/ftp_upload/53533/53533fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53533/53533fig2.jpg" /> 图 2.使用 注射器塞子按通过70mm的细胞过滤剩余的红髓。（A）脾脏后剩余的红色纸浆我<html> š加工成单细胞悬浮液，并通过70毫米细胞过滤通过。 （B）一种3毫升注射器的止动部分用于轻轻按通过细胞过滤剩余的红髓。 （ 三）用注射器之后，应该几乎没有红髓留在细胞过滤。 <a href="https://www.jove.com/files/ftp_upload/53533/53533fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53533/53533fig3.jpg" /> 图3. ACK裂解红血细胞。从至ACK裂解之前单个小鼠脾脏中产生的单细胞悬浮液（A）中离心。 （B）中的红血细胞ACK裂解后，将细胞沉淀为白色。获得="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图4" src="/files/ftp_upload/53533/53533fig4.jpg" /> 图 协议的代表性 的成果 。（A）选通方案用于分析CD4 + T淋巴细胞的染色质状态。 （B和C）的T细胞不处理或用1毫克/毫升的可溶性抗CD3抗体3小时（一式三份）激活。然后将细胞通过流式细胞术分析，以确定在CD4 +细胞（B）和CD8 +细胞（C）的染色质凝聚。数据是平均值±H3K4me1染色的平均荧光强度的标准偏差。 * p &lt;0.001（学生t-检验 ） <a href="https://www.jove.com/files/ftp_upload/53533/53533fig4large.jpg" target="_blank">请点击ħ</a>ERE查看该图的放大版本。 <img alt="表格1" src="/files/ftp_upload/53533/53533table1.jpg" /> 表1:故障排除指南的快速参考常见问题和可能的解决方案。

Watch this Scientific Journal Video about The Use of Flow Cytometry to Assess the State of Chromatin in T Cells at JoVE.com

The Use of Flow Cytometry to Assess the State of Chromatin in T Cells

流式细胞仪用来评估染色质的T细胞国家

流式细胞仪可用于T细胞内的染色质评估的状态。该协议允许科学家在T细胞活化的增加的荧光组蛋白H3的抗体的平均荧光强度（MFI）证明解释染色质解聚的证据

During a proper immune response, quiescent T cells become activated upon antigen presentation to their antigen-specific T cell receptor. This leads to ...

the-use-of-flow-cytometry-to-assess-the-state-of-chromatin-in-t-cells

Research

JoVE Journal

Immunology and Infection

16.8K 次观看.  Furman University. 流式细胞仪可用于T细胞内的染色质评估的状态。该协议允许科学家在T细胞活化的增加的荧光组蛋白H3的抗体的平均荧光强度（MFI）证明解释染色质解聚的证据

视频: The Use of Flow Cytometry to Assess the State of Chromatin in T Cells

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 ...

科研

免疫与感染

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 ...

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

评估视网膜小胶质细胞吞噬功能<em&gt;在体内</em&gt;使用基于流式细胞术流量测定

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.

干细胞衍生的抗原特异性调节性T细胞对自身免疫的发展

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal of immunosuppression. The mixed leukocyte reaction (MLR), limiting dilution assays, and trans-vivo delayed-type hypersensitivity (DTH) assay have all been applied to this question, but these methods have limited predictive ability and/or significant practical limitations that reduce their usefulness. Imaging flow cytometry is a technique that combines the multiparametric quantitative powers of flow cytometry with the imaging capabilities of fluorescent microscopy. We recently made use of an imaging flow cytometry approach to define the proportion of recipient T cells capable of forming mature immune synapses with donor antigen-presenting cells (APCs). Using a well-characterized mouse heart transplant model, we have shown that the frequency of in vitro immune synapses among T-APC membrane contact events strongly predicted allograft outcome in rejection, tolerance, and a situation where transplant survival depends on induced regulatory T cells. The frequency of T-APC contacts increased with T cells from mice during acute rejection and decreased with T cells from mice rendered unresponsive to alloantigen. The addition of regulatory T cells to the in vitro system reduced prolonged T-APC contacts. Critically, this effect was also seen with human polyclonally expanded, naturally occurring regulatory T cells, which are known to control the rejection of human tissues in humanized mouse models. Further development of this approach may allow for a deeper characterization of the alloreactive T-cell compartment in transplant recipients. In the future, further development and evaluation of this method using human cells may form the basis for assays used to select patients for immunosuppression minimization, and it can be used to measure the impact of tolerogenic therapies in the clinic.

This paper describes a method for measuring alloreactivity in a mixed population of T cells using imaging flow cytometry.

使用成像流式细胞仪T细胞同种异体反应性测定

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal ...

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 ...

Multicolor Flow Cytometry-based Quantification of Mitochondria and Lysosomes in T Cells

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular components responsible for supplying cell energy; however, excess mitochondria also produce reactive oxygen species (ROS) that could cause cell death. Therefore, the number of mitochondria must constantly be adjusted to fit the needs of the cells. This dynamic regulation is achieved in part through the function of lysosomes that remove surplus/damaged organelles and macromolecules. Hence, cellular mitochondrial and lysosomal contents are key indicators to evaluate the metabolic adjustment of cells. With the development of probes for organelles, well-characterized lysosome or mitochondria-specific dyes have become available in various formats to label cellular lysosomes and mitochondria. Multicolor flow cytometry is a common tool to profile cell phenotypes, and has the capability to be integrated with other assays. Here, we present a detailed protocol of how to combine organelle-specific dyes with surface markers staining to measure the amount of lysosomes and mitochondria in different T cell populations on a flow cytometer.

This article illustrates a powerful method to quantify mitochondria or lysosomes in living cells. The combination of lysosome- or mitochondria-specific dyes with fluorescently conjugated antibodies against surface markers allows the quantification of these organelles in mixed cell populations, like primary cells harvested from tissue samples, by using multicolor flow cytometry.

基于多色流式细胞仪的 t 细胞线粒体和溶酶体的定量

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular ...

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 ...

Isolation of Mouse Kidney-Resident CD8+ T cells for Flow Cytometry Analysis

Tissue-resident memory T cell (TRM) is a rapidly expanding field of immunology research. Isolating T cells from various non-lymphoid tissues is one of the key steps to investigate TRMs. There are slight variations in lymphocyte isolation protocols for different organs. Kidney is an essential non-lymphoid organ with numerous immune cell infiltration especially after pathogen exposure or autoimmune activation. In recent years, multiple labs including our own have started characterizing kidney resident CD8+ T cells in various physiological and pathological settings in both mouse and human. Due to the abundance of T lymphocytes, kidney represents an attractive model organ to study TRMs in non-mucosal or non-barrier tissues. Here, we will describe a protocol commonly used in TRM-focused labs to isolate CD8+ T cells from mouse kidneys following systemic viral infection. Briefly, using an acute lymphocytic choriomeningitis virus (LCMV) infection model in C57BL/6 mice, we demonstrate intravascular CD8+ T cell labeling, enzymatic digestion, and density gradient centrifugation to isolate and enrich lymphocytes from mouse kidneys to make samples ready for the subsequent flow cytometry analysis.

Isolation of Mouse Kidney-Resident CD8+ T cells for Flow Cytometry Analysis

Following viral infection, kidney harbors a relatively large number of CD8+ T cells and offers an opportunity to study non-mucosal TRM cells. Here, we describe a protocol to isolate mouse kidney lymphocytes for flow cytometry analysis.

小鼠肾-居民CD8+T 细胞的分离，用于流细胞测量分析

Tissue-resident memory T cell (TRM) is a rapidly expanding field of immunology research. Isolating T cells from various non-lymphoid tissues is one of the ...

Analysis of SAMHD1 Restriction by Flow Cytometry in Human Myeloid U937 Cells

Sterile &#945;-motif/histidine-aspartate domain-containing protein 1 (SAMHD1) inhibits replication of HIV-1 in quiescent myeloid cells. U937 cells are widely used as a convenient cell system for analyzing SAMHD1 activity due to a low level of SAMHD1 RNA expression, leading to undetectable endogenous protein expression. Based on similar assays developed in the Stoye laboratory to characterize other retroviral restriction factors, the Bishop lab developed a two-color restriction assay to analyze SAMHD1 in U937 cells. Murine Leukaemia Virus-like particles expressing SAMHD1, alongside YFP expressed from an IRES, are used to transduce U937 cells. Cells are then treated with phorbol myristate acetate to induce differentiation to a quiescent phenotype. Following differentiation, cells are infected with HIV-1 virus-like particles expressing a fluorescent reporter. After 48 h, cells are harvested and analyzed by flow cytometry. The proportion of HIV-infected cells in the SAMHD1-expressing population is compared to that in internal control cells lacking SAMHD1. This comparison reveals a restriction ratio. SAMHD1 expression leads to a five-fold reduction in HIV infection, corresponding to a restriction ratio of 0.2. Our recent substitution of RFP for the original GFP as the reporter gene for HIV infection has facilitated flow cytometry analysis.
This assay has been successfully used to characterize the effect of amino acid substitutions on SAMHD1 restriction by transducing with viruses encoding altered SAMHD1 proteins, derived from site-directed mutagenesis of the expression vector. For example, the catalytic site substitutions HD206-7AA show a restriction phenotype of 1, indicating a loss of restriction activity. Equally, the susceptibility of different tester viruses can be determined. The assay can be further adapted to incorporate the effect of differentiation status, metabolic status, and SAMHD1 modifiers to better understand the relationship between SAMHD1, cell metabolic state, and viral restriction.

Described here is an established method to determine the extent of HIV-1 restriction by the cellular inhibitory protein SAMHD1. Human myeloid lineage U937 cells are transduced with a SAMHD1 expression vector co-expressing YFP, differentiated and then challenged with HIV-RFP. The level of restriction is determined by flow cytometry analysis.

流式细胞术分析人髓系U937细胞中SAMHD1限制性

Sterile &#945;-motif/histidine-aspartate domain-containing protein 1 (SAMHD1) inhibits replication of HIV-1 in quiescent myeloid cells. U937 cells are ...