Name: the use of flow cytometry to assess the state of chromatin in t cells
Uploaded: 2015-12-17T14:00:00.000+00:00
Duration: 11 min 1 s
Description: Watch this Scientific Journal Video about The Use of Flow Cytometry to Assess the State of Chromatin in T Cells at JoVE.com

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><tbody><tr><td>100 mm tissue culture dish</td><td>BD Biosciences</td><td>353003</td><td></td></tr><tr><td>Precleaned frosted microscope slides</td><td>Fisher Scientific</td><td>12-550-343</td><td></td></tr><tr><td>Sterile cell strainer, 70 mm nylon mesh</td><td>Fisher Scientific</td><td>22363548</td><td></td></tr><tr><td>3 ml syringe, Luer-Lok tip</td><td>BD</td><td>309585</td><td></td></tr><tr><td>Falcon 50 ml polypropylene conical tube</td><td>Corning</td><td>352098</td><td></td></tr><tr><td>LIVE/DEAD fixable red dead cell stain kit</td><td>Life Technologies</td><td>L23102</td><td>Life Technologies sells versions of this kit with different colors</td></tr><tr><td>Fc Block</td><td>BD Biosciences</td><td>553142</td><td></td></tr><tr><td>16% paraformaldehyde</td><td>Electron Microscopy Sciences</td><td>15710</td><td></td></tr><tr><td>Triton X-100</td><td>Fisher Scientific</td><td>BP151-100</td><td></td></tr><tr><td>Normal rabbit serum</td><td>Sigma-Aldrich</td><td>R9133</td><td></td></tr><tr><td>Anti-H3K4me1 antibody</td><td>Abcam</td><td>ab8895</td><td></td></tr><tr><td>R-Phycoerythrin conjugation kit</td><td>Abcam</td><td>ab102919</td><td>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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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

Universidad San Sebastian

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

Examination of Thymic Positive and Negative Selection by Flow Cytometry

A healthy immune system requires that T cells respond to foreign antigens while remaining tolerant to self-antigens. Random rearrangement of the T cell receptor (TCR) &alpha; and &beta; loci generates a T cell repertoire with vast diversity in antigen specificity, both to self and foreign. Selection of the repertoire during development in the thymus is critical for generating safe and useful T cells. Defects in thymic selection contribute to the development of autoimmune and immunodeficiency disorders1-4. 
T cell progenitors enter the thymus as double negative (DN) thymocytes that do not express CD4 or CD8 co-receptors. Expression of the &alpha;&beta;TCR and both co-receptors occurs at the double positive (DP) stage. Interaction of the &alpha;&beta;TCR with self-peptide-MHC (pMHC) presented by thymic cells determines the fate of the DP thymocyte. High affinity interactions lead to negative selection and elimination of self-reactive thymocytes. Low affinity interactions result in positive selection and development of CD4 or CD8 single positive (SP) T cells capable of recognizing foreign antigens presented by self-MHC5.
Positive selection can be studied in mice with a polyclonal (wildtype) TCR repertoire by observing the generation of mature T cells. However, they are not ideal for the study of negative selection, which involves deletion of small antigen-specific populations. Many model systems have been used to study negative selection but vary in their ability to recapitulate physiological events6. For example, in vitro stimulation of thymocytes lacks the thymic environment that is intimately involved in selection, while administration of exogenous antigen can lead to non-specific deletion of thymocytes7-9. Currently, the best tools for studying in vivo negative selection are mice that express a transgenic TCR specific for endogenous self-antigen. However, many classical TCR transgenic models are characterized by premature expression of the transgenic TCR&alpha; chain at the DN stage, resulting in premature negative selection. Our lab has developed the HYcd4 model, in which the transgenic HY TCR&alpha; is conditionally expressed at the DP stage, allowing negative selection to occur during the DP to SP transition as occurs in wildtype mice10.
Here, we describe a flow cytometry-based protocol to examine thymic positive and negative selection in the HYcd4 mouse model. While negative selection in HYcd4 mice is highly physiological, these methods can also be applied to other TCR transgenic models. We will also present general strategies for analyzing positive selection in a polyclonal repertoire applicable to any genetically manipulated mice.

We present a flow cytometry-based method to examine T cell development in vivo using genetically manipulated mice on a wildtype or T cell receptor transgenic background.

考试流式细胞仪检测胸腺阳性和阴性选择

A healthy immune system requires that T cells respond to foreign  antigens while remaining tolerant to self-antigens. Random rearrangement of the  T cell ...

科研

免疫与感染

Rapid and Robust Analysis of Cellular and Molecular Polarization Induced by Chemokine Signaling

Cells respond to chemokine stimulation by losing their round shape in a process called polarization, and by altering the subcellular localization of many proteins. Classic imaging techniques have been used to study these phenomena. However, they required the manual acquisition of many cells followed by time consuming quantification of the morphology and the co-localization of the staining of tens of cells. Here, a rapid and powerful method is described to study these phenomena on samples consisting of several thousands of cells using an imaging flow cytometry technology that combines the advantages of a microscope with those of a cytometer. Using T lymphocytes stimulated with CCL19 and staining for MHC Class I molecules and filamentous actin, a gating strategy is presented to measure simultaneously the degree of shape alterations and the extent of co-localization of markers that are affected by CCL19 signaling. Moreover, this gating strategy allowed us to observe the segregation of filamentous actin (at the front) and phosphorylated Ezrin-Radixin-Moesin (phospho-ERM) proteins (at the rear) in polarized T cells after CXCL12 stimulation. This technique was also useful to observe the blocking effect on polarization of two different elements: inhibition of actin polymerization by a pharmacological inhibitor and expression of mutants of the Par6/atypical PKC signaling pathway. Thus, evidence is shown that this technique is useful to analyze both morphological alterations and protein redistributions.

Chemokine signaling elicits marked alterations of cellular morphology and some important redistributions of intracellular proteins. Here, a rapid and detailed protocol is provided to study these events.

细胞与分子极化的快速和强大的分析趋化因子诱导信号

Cells respond to chemokine stimulation by losing their round shape in a process called polarization, and by altering the subcellular localization of many ...

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 ...

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 ...

Flow Cytometric Characterization of Murine B Cell Development

Extensive studies have characterized the development and differentiation of murine B cells in secondary lymphoid organs. Antibodies secreted by B cells have been isolated and developed into well-established therapeutics. Validation of murine B cell development, in the context of autoimmune prone mice, or in mice with modified immune systems, is a crucial component of developing or testing therapeutic agents in mice and is an appropriate use of flow cytometry. Well established B cell flow cytometric parameters can be used to evaluate B cell development in the murine peritoneum, bone marrow, and spleen, but a number of best practices must be adhered to. In addition, flow cytometric analysis of B cell compartments should also complement additional readouts of B cell development. Data generated using this technique can further our understanding of wild type, autoimmune prone mouse models as well as humanized mice that can be used to generate antibody or antibody-like molecules as therapeutics.

We describe herein a simple analysis of the heterogeneity of the murine immune B cell compartment in the peritoneum, spleen, and bone marrow tissues by flow cytometry. The protocol can be adapted and extended to other mouse tissues.

小鼠B细胞发育的流式细胞学表征

Extensive studies have characterized the development and differentiation of murine B cells in secondary lymphoid organs. Antibodies secreted by B cells ...

A DNA/Ki67-Based Flow Cytometry Assay for Cell Cycle Analysis of Antigen-Specific CD8 T Cells in Vaccinated Mice

The cell cycle of antigen-specific T cells in vivo has been examined by using a few methods, all of which possess some limitations. Bromodeoxyuridine (BrdU) marks cells that are in or recently completed S-phase, and carboxyfluorescein succinimidyl ester (CFSE) detects daughter cells after division. However, these dyes do not allow identification of the cell cycle phase at the time of analysis. An alternative approach is to exploit Ki67, a marker that is highly expressed by cells in all phases of the cell cycle except the quiescent phase G0. Unfortunately, Ki67 does not allow further differentiation as it does not separate cells in S-phase that are committed to mitosis from those in G1 that can remain in this phase, proceed into cycling, or move into G0.
Here, we describe a flow cytometric method for capturing a &#34;snapshot&#34; of T cells in different cell cycle phases in mouse secondary lymphoid organs. The method combines Ki67 and DNA staining with major histocompatibility complex (MHC)-peptide-multimer staining and an innovative gating strategy, allowing us to successfully differentiate between antigen-specific CD8 T cells in G0, in G1 and in S-G2/M phases of the cell cycle in the spleen&#160;and draining lymph nodes of mice after vaccination with viral vectors carrying the model antigen gag of human immunodeficiency virus (HIV)-1.
Critical steps of the method were the choice of the DNA dye and the gating strategy to increase the assay sensitivity and to include highly activated/proliferating antigen-specific T cells that would have been missed by current criteria of analysis. The DNA dye, Hoechst 33342, enabled us to obtain a high-quality discrimination&#160;of the G0/G1 and G2/M DNA peaks, while preserving membrane and intracellular staining. The method has great potential to increase knowledge about T cell response in vivo and to improve immuno-monitoring analysis.

Clonal expansion is a key feature of antigen-specific T cell response. However, the cell cycle of antigen-responding T cells has been poorly investigated, partly because of technical limitations. We describe a flow cytometric method to analyze clonally expanding antigen-specific CD8 T cells in spleen and lymph nodes of vaccinated mice.

一种基于DNA/Ki67的流式细胞术测定法，用于接种疫苗的小鼠抗原特异性CD8 T细胞的细胞周期分析

The cell cycle of antigen-specific T cells in vivo has been examined by using a few methods, all of which possess some limitations. Bromodeoxyuridine ...

Temporal Tracking of Cell Cycle Progression Using Flow Cytometry without the Need for Synchronization

This protocol describes a method to permit the tracking of cells through the cell cycle without requiring the cells to be synchronized. Achieving cell synchronization can be difficult for many cell systems. Standard practice is to block cell cycle progression at a specific stage and then release the accumulated cells producing a wave of cells progressing through the cycle in unison. However, some cell types find this block toxic resulting in abnormal cell cycling, or even mass death. Bromodeoxyuridine (BrdU) uptake can be used to track the cell cycle stage of individual cells. Cells incorporate this synthetic thymidine analog, while synthesizing new DNA during S phase. By providing BrdU for a brief period it is possible to mark a pool of cells that were in S phase while the BrdU was present. These cells can then be tracked through the remainder of the cell cycle and into the next round of replication, permitting the duration of the cell cycle phases to be determined without the need to induce a potentially toxic cell cycle block. It is also possible to determine and correlate the expression of both internal and external proteins during subsequent stages of the cell cycle. These can be used to further refine the assignment of cell cycle stage or assess effects on other cellular functions such as checkpoint activation or cell death.

This protocol describes the use of bromodeoxyuridine (BrdU) uptake to permit the temporal tracking of cells that were in S phase at a specific point in time. Addition of DNA dyes and antibody labeling facilitates detailed analysis of the fate of the S phase cells at later times.

细胞周期进程的时间跟踪使用流式细胞仪，无需同步

This protocol describes a method to permit the tracking of cells through the cell cycle without requiring the cells to be synchronized. Achieving cell ...

生物学

使用流式细胞术进行小鼠脑组蛋白修饰定量

Quantification of Global Histone Post Translational Modifications Using Intranuclear Flow Cytometry in Isolated Mouse Brain Microglia

Gene expression control occurs partially by modifications in chromatin structure, including the addition and removal of posttranslational modifications to histone tails. Histone post-translational modifications (HPTMs) can either facilitate gene expression or repression. For example, acetylation of histone tail lysine residues neutralizes the positive charge and reduces interactions between the tail and negatively charged DNA. The decrease in histone tail-DNA interactions results in increased accessibility of the underlying DNA, allowing for increased transcription factor access. The acetylation mark also serves as a recognition site for bromodomain-containing transcriptional activators, together resulting in enhanced gene expression. Histone marks can be dynamically regulated during cell differentiation and in response to different cellular environments and stimuli. While next-generation sequencing approaches have begun to characterize genomic locations for individual histone modifications, only one modification can be examined concurrently. Given that there are hundreds of different HPTMs, we have developed a high throughput, quantitative measure of global HPTMs that can be used to screen histone modifications prior to conducting more extensive genome sequencing approaches. This protocol describes a flow cytometry-based method to detect global HPTMs and can be conducted using cells in culture or isolated cells from in vivo tissues. We present example data from isolated mouse brain microglia to demonstrate the sensitivity of the assay to detect global shifts in HPTMs in response to a bacteria-derived immune stimulus (lipopolysaccharide). This protocol allows for the rapid and quantitative assessment of HPTMs and can be applied to any transcriptional or epigenetic regulator that can be detected by an antibody.

作者聚焦:基因表达调控研究的增强

This work describes a protocol for the quantification of global histone modifications using intranuclear flow cytometry in isolated brain microglia. The work also contains the microglia isolation protocol that was used for data collection.

使用核内流式细胞术定量分离小鼠脑小胶质细胞中的整体组蛋白翻译后修饰

Gene expression control occurs partially by modifications in chromatin structure, including the addition and removal of posttranslational modifications to ...

流式细胞仪用来评估染色质的T细胞国家

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考试流式细胞仪检测胸腺阳性和阴性选择

细胞与分子极化的快速和强大的分析趋化因子诱导信号

使用成像流式细胞仪T细胞同种异体反应性测定

基于多色流式细胞仪的 t 细胞线粒体和溶酶体的定量

小鼠B细胞发育的流式细胞学表征

一种基于DNA/Ki67的流式细胞术测定法，用于接种疫苗的小鼠抗原特异性CD8 T细胞的细胞周期分析

细胞周期进程的时间跟踪使用流式细胞仪，无需同步

使用核内流式细胞术定量分离小鼠脑小胶质细胞中的整体组蛋白翻译后修饰

使用核内流式细胞术定量分离小鼠脑小胶质细胞中的整体组蛋白翻译后修饰

使用核内流式细胞术定量分离小鼠脑小胶质细胞中的整体组蛋白翻译后修饰