这项工作是由国家批卫生研究院：PO1 HL55798（吉隆坡）和美国心脏协会的科学家开发授予0525532U（EG）的支持。

1。分离小鼠主动脉机构IACUC委员会批准的程序是必需的工作与小鼠。 <ol><li>准备加入肝素钠1000个单位，50毫升PBS和反相混合管heparinzed PBS。准备一个空的收集管的血液和集合管中，PBS为每个要收集的主动脉。所有管置于冰上。 </li><li> Heparinize一个注射器吸取血（1000 U / ml的肝素钠0.1毫升），准备手术工具（两对弯钳，一双解剖剪刀，和一双的microshears），并为解剖解剖阶段。 </li><li>安乐死鼠标使用二氧化碳，在核准的商会，美国国立卫生研究院和机构IACUC委员会关于啮齿类动物安乐死的政策。将鼠标解剖阶段之前检查的有效性。 </li><li>简要地用70％乙醇浸泡鼠标和鼠标固定清扫阶段。鼠标通过心脏穿刺吸取血。 </li><li>打开腹部和胸部腔。使用一个25克针的10毫升注射器，完全用PBS含有肝素的2％，彻底清除血液从心脏穿刺血管的血管灌注。确保没有主动脉组织内的血液。应进行灌注，慢慢地与小的压力，以确保所有斑块在血管壁保持不变。 </li><li>解剖并取出内脏器官，泌尿生殖系统器官，膈肌和脾，使肾脏，心脏和主动脉完好无损。 </li><li>仔细解剖脂肪组织和腹主动脉旁淋巴结离主动脉，使主动脉和外膜完好。收集包括整个主动脉主动脉弓，升，降，胸，腹部分。将收集管中，用PBS的离体主动脉。在隔离过程中，尽量保持船只的湿润。 </li></ol> 2。单细胞悬液的制备<ol><li>创建一个1X主动脉分解酶原液（ADES）（125 U / mL胶原酶型XI，透明质酸类型60 U / ml的1 - S，60 U / ml的DNA酶我，和我，在2.5毫升450 U / mL胶原酶类型PBS中，修改加尔金娜等7）。所有的酶都是由Sigma - Aldrich公司。将股票酶液在冰上。 </li><li>从集合中删除包含主动脉管的PBS。加入2.5毫升的1X的ADE每个主动脉。切成小块的主动脉，以方便酶消化或保持整体主动脉不变。的主动脉孵育1小时1X酶的解决方案，在37 ° C（慢摇是可选的）。 </li><li> 1小时的潜伏期后，准备从消化主动脉主动脉除了剪切和70微米的细胞过滤器通过流式细胞仪进入5毫升聚丙烯管（BD猎鹰）单细胞悬液。颗粒离心细胞（400xg，5分钟，4℃）。 </li><li>重悬细胞，流式细胞仪缓冲液（PBS，用1％BSA和0.05％NaN 的 3补充）1毫升和确定多少细胞是目前使用台盼蓝，血球，和光镜下的细胞悬浮液在主动脉。消化后获得的细胞总数将取决于对小鼠的年龄和饮食或动脉粥样硬化的严重程度。 </li></ol> 3。流式细胞仪染色<ol><li>标签适当数量的新流式细胞仪管。在一般情况下，流式细胞仪检测实验应该有一个非彩色控制管，单一颜色的管，适当的荧光减一控制（FMO） 的 8，相应的同型对照，实验管设置。既然有可以离体主动脉白细胞数量有限，脾脏白细胞可用于执行单一控制染色。 </li><li>我们用一个标准的流式细胞仪协议染色主动脉细胞悬液。简单地说，转移0.5 - 1 × 10 6细胞成流式细胞仪管（S）的主动脉细胞悬液等分。新增离心1毫升流式细胞仪缓冲液和颗粒细胞。调迁从颗粒细胞中取出上清液。 </li><li>准备实验管，荧光减一管，和同型管FC块解决方案和抗体鸡尾酒。 FC流式细胞仪缓冲块（14.2μg/ml，克隆2.4G2）添加100μL管和手指轻弹，或轻轻涡旋悬浮细胞。 15-20分钟，在室温下孵育样本。 </li><li>准备抗体染色，染色亚型或鱼类统营处控制染色鸡尾酒。初步滴定实验确定最佳的抗体浓度。在FC块的存在，增加抗体鸡尾酒（100ul/0.5-1.0x10 6细胞）的样品管。孵育20-30分钟，在4 ° C黑暗环境中。 </li><li>流式细胞仪缓冲1ml到每个管，涡旋混合，并沉淀，离心细胞。重复此过程，更多的时间。 </li><li>倒出上清液和重悬300μL的2％的煤灰颗粒细胞。流式细胞仪上运行的样本。 <ul><li>注：通常情况下的抗CD45抗体（常见的白细胞抗原标记）被添加到所有的样本中，以门 CD45 + 白细胞后7此外，CD45的鱼类统营处和同型对照应该是最初使用放置CD45 +白细胞门。 CD45的表达，可以改变其中白细胞。 </li><li>注：为了表达表面抗原或低的事件抗原检测密度低，使用“明亮的荧光，如R -藻红蛋白（PE）或变藻蓝蛋白（APC）”。此外，罕见的事件可以被检测到几个匹配的主动脉汇集在一起​​;然而，如果使用超过一百万个细胞，每个测试使用的抗体量应增加比例。 </li><li>注：为了确保有最小中分离出来的主动脉血液污染，因此在主动脉细胞悬液，在一些额外的染色实验TER - 119，红细胞（RBC）的表达抗原执行。红细胞数量在血液中白细胞（WBC）的大约10 × 10 6细胞/μL8x10的3细胞/μL。主动脉样品的TER - 119表达的基础上，血源性样品中的白血细胞的比例可以计算的。通常情况下，我们已经在主动脉样品的血源性白细胞小于0.02％。 （图） </li><li>注意：由于酶处理可能会影响表面抗原的表达，酶消化的阻力应为利益的抗原决定的。简单地说，周围淋巴结（PLN）或脾小块孵育在37 ° C或1小时没有酶的鸡尾酒1小时后，抗​​原的表达，流式细胞仪检测。酶的鸡尾酒有多个表面抗原（ 图2）7没有影响。在另一些情况下，酶处理抗原表达的一个重大损失。这个问题可以规避使用替代细胞标记。 </li><li>注：活/死细胞活力染色可以执行第4步后，如果需要的话。通常使用活/死可以解决的死细胞染色试剂盒（Invitrogen公司，分子探针）。要活/死染料染色的细胞，创建一个加入PBS和涡1毫升重新溶解活/死染料加入1μl混合1X活/死的解决方案。加入100-200μl1X活/死染料的生活/死亡单，鸡尾酒，鱼类统营处和同型对照样品。孵育的样品在室温为10分钟，在黑暗中的染料。单一的控制管活/死应在56℃孵育° C黑暗的为10分钟，以杀死细胞热休克环境中。洗净用1毫升PBS和沉淀，离心细胞的细胞。在第5步恢复协议。 </li><li>注：细胞内的抗原和细胞因子的细胞内染色与此协议兼容。简单来说，细胞内染色可以使用BD Pharmingen公司，eBioscience公司，或Caltag实验室的细胞固定/通透试剂 - 根据感兴趣的抗原。为了修复和通透细胞内染色的细胞，固定/通透步骤执行后，制造商的指示后，细胞染色步骤（第3条第5步）。细胞内的洗涤步骤，在第6步（第3条第6步）恢复我们的协议。 </li></ul></li></ol> 4。流量隔离周围的外膜和血管壁的流式细胞仪分析由于白细胞可以移植到主动脉外膜以及主动脉壁内动脉粥样硬化斑块外膜和主动脉白细胞流式细胞仪研究开发一种隔离和执行这两个解剖部位流式细胞仪的协议9。简要之前，整个主动脉消化与主动脉外膜部分被消化和删除其余船只ADES（第2，第2步）。一旦外膜被删除，并预留，其余主动脉消化的ADE解放白细胞从血管壁。 <ul><li> 主动脉外膜隔离协议 </li></ul><ol><li>以下1X的ADE（第2条第1步）的准备，准备为2.5毫升/ 1X主动脉外膜消化酶液（AADES）（781.25 ü胶原酶II和14.0625 ü弹性蛋白酶2.5mls PBS（沃辛顿生化公司，新泽西州莱克伍德的主动脉上，直到用冰））9将原液。 </li><li>从集合中删除包含主动脉管的PBS。加入2.5毫升的1X AADES每个主动脉。不要切成小块的主动脉。 1X外膜酶液的主动脉孵育10-20分钟，在37 ° C。 </li><li> 10-20分钟的消化之后，部分消化的主动脉1X AADES转移到用新鲜PBS的培养皿中。非常仔细，使用两弯钳，剥离外膜层远离FROM主动脉作为一个单元。 <ul><li>注意：较长的消化时间，使其更易于去除外膜;但是，如果外膜消化，它会撕裂。 </li></ul></li><li>当外膜被完全删除，转移的外膜和主动脉管单独的流式细胞仪。新增的PBS 1ml至外膜管，管置于冰上。 1X主动脉分解酶液加入2.5毫升的主动脉管40分钟，主动脉，并在37 ° C。 <ul><li>注：为了促进消化酶，主动脉可分为较小的部分，在这一点上最多。 </li></ul></li><li> 40分钟的消化之后，放置在冰主动脉管，剪切的主动脉和外膜分开，通过流式细胞仪进入5毫升聚丙烯管（BD猎鹰）70微米的细胞过滤器，并准备从主动脉和外膜单细胞悬液。颗粒离心细胞（400xg，5分钟，4℃）。返回到第2条第4步和恢复的协议。 </li></ol> 5。代表性的成果在这里，我们目前的数字表明流式细胞仪染色分析免疫成分的全主动脉，主动脉血管壁和周围的主动脉外膜。首先，我们展示了一个有代表性的流式细胞仪图显示一个TER - 119全血和离体主动脉细胞悬液（ 图1）染色。 TER - 119阳性红细胞占18％主动脉细胞悬液，这表明，只有0.014％，从主动脉细胞悬浮液中分离出的细胞是血源性细胞。这是一个重要的控制实验，清楚地表明，消化船只，但不循环外周血为主动脉细胞悬液分析的最白细胞的来源。此外，为验证方法，我们评估主动脉解离脾细胞表面抗原的酶鸡尾酒（ 图 2）的影响。酶鸡尾酒几个抗原，包括CD45，CD19 +，CD3 +，TCRαβ，TCRγδ和其他几个表面抗原的表达没有影响。 CD45的结合活/死的可行性染料和适当的同型对照染色用于检测和分闸的主要群体的利益，并排除在分析过程中的细胞碎片。在图3，现场主动脉CD45 +白细胞干扰素的百分比来确定的门 Υ+ T细胞内两个年轻的 APOE汇集主动脉- / -小鼠。为了强调这种方法的通用性，使用门控图3中提出的计划， 我们目前在图代表从两个ApoE基因的细胞内染色CD68 +细胞CD11b +巨噬细胞和IFNγ+TCRαβ+细胞 - / -小鼠的西方饮食喂养12周。正如预期的那样在西方饮食喂养ApoE基因 ， - / -主动脉，主动脉内的大多数白细胞，巨噬细胞（40％CD68 +细胞CD11b +）或其他髓系细胞（细胞CD11b + CD68低和CD11b + CD68 -）。此外，Th1细胞组成的TCRαβT细胞（ 图4）浸润主动脉的主要部分。由于主动脉白细胞和白细胞亚群的整体比例取决于鼠标的年龄和动脉粥样硬化的严重程度不同，关心的重大人口的最佳浇注战略应凭经验确定为了证明隔离流式细胞仪染色的主动脉外膜的可行性，我们目前代表CD45 +主动脉和外膜细胞悬浮液中白细胞染色（ 图 5 ）。简单地说，三岁的ApoE基因 - / -小鼠主动脉被消化和汇集如上所述（第2和第4条）。浸润的CD45 +白细胞检测在外膜的细胞悬液（18％），其余主动脉（11％）的细胞悬液。 <img src="/files/ftp_upload/2848/2848fig1.jpg" alt="图1"/> 图1。TER - 119染色消化主动脉细胞悬液。主动脉灌注心脏穿刺用PBS含2％肝素。然后在主动脉消化在37 ° C，1小时的酶鸡尾酒主动脉细胞悬液和血液样本（作为阳性对照）与反TER - 119 - PE ABS染色​​和流式细胞仪分析。 TER - 119阳性红细胞占18％主动脉细胞悬浮表明，只有0.014％，从主动脉隔离所有白细胞可能是血源性白细胞中的所有细胞。 <img src="/files/ftp_upload/2848/2848fig2.jpg" alt="图2"/> 图2。酶鸡尾酒疗法CD45（顶部）或CD19（底部）对淋巴细胞的表达没有影响 。 </stronG&gt;从未经处理的（A，B）和酶鸡尾酒（C，D）处理细胞悬液LN获得，并与APC - Cy7标记的抗CD45和APC标记的抗CD19的单克隆抗体染色。 （A，C）的数字表示在R1门CD45 +的白细胞的百分比。 （B，D）的数字表示的CD45 + / CD19 +淋巴细胞的百分比。资料是对CD45 +白细胞门。 <img src="/files/ftp_upload/2848/2848fig3.jpg" alt="图3"/> 图3浇注战略分析主动脉白细胞中分离出两个主动脉和汇集从容易发生动脉粥样硬化ApoE基因 - / -小鼠和主动脉细胞悬液的制备如上所述。细胞悬浮液染色CD45（PerCP），TCRαβ（FITC），γ干扰素（eFluor 450），和活/死的Aqua，并使用一个Cytek DXP的8色升级BD流式细胞仪刀魂分析。简言之，CD45 +白细胞门（二）进一步分析（CF）。从死细胞活/死的Aqua染色（D）和FSC的阴谋（五）为基础的分析。现场主动脉白细胞，然后TCRαβ和IFNγ表达（F）的研究。 <img src="/files/ftp_upload/2848/2848fig4.jpg" alt="图4"/> 图4。细胞内的抗原和细胞因子的细胞内染色细胞悬液的制备从一个整体 的ApoE基因 - / -主动脉和脾。在主动脉（A）和脾（B，C）的细胞悬浮液染色CD45，CD11b的，和CD68或同型对照使用BD Cytofix / Cytoperm™试剂盒（BD Biosciences公司）。细胞上的CD45 +白细胞gated和碎片被排除在外的基础上正向和侧向散射配置文件。细胞内IFNγ染色，主动脉和脾细胞悬浮培养与高尔基停止，PMA和离子霉素彗星为辅的RPMI 1640五小时，如前面描述的。刺激单主动脉（D）和脾脏（E，F）的细胞悬浮液随后，TCRαβCD45（PerCP）（FITC），CD3（APC - Cy7）染色，过死的Aqua和IFNγ（eFluor 450，E）或亚型（IgG1的eF450，女）。 T细胞（DF）的门生活CD45 + CD3 +白细胞（CD45 +活/死的Aqua）和TCRαβ和IFNγ检查。 <img src="/files/ftp_upload/2848/2848fig5.jpg" alt="图5"/> 图5。孤立的小鼠主动脉外膜和主动脉血管壁的代表形象代表流式细胞仪计数器情节演示的 CD45 +存在T细胞外膜和主动脉壁岁 ApoE基因- / -小鼠。 SSC的侧向散射。要消除碎片和坏死组织的自体荧光，情节对FSC&gt; 750门。为了避免额外的自体荧光的大门也被设置为南南合作&lt;3500 &lt;3500，FSC双峰。百分比表示CD45 +细胞的大门。

<ol>
	<li>Lusis, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atherosclerosis.">Atherosclerosis.</a> Nature. 407, 233-2341 (2000).</li><li>Galkina, E., Ley, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+and+inflammatory+mechanisms+of+atherosclerosis.">Immune and inflammatory mechanisms of atherosclerosis.</a> Annu Rev Immunol. 27, 165-197 (2009).</li><li>Hansson, G. K., Libby, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+immune+response+in+atherosclerosis:+a+double-edged+sword.">The immune response in atherosclerosis: a double-edged sword.</a> Nat Rev Immunol. 6, 508-519 (2006).</li><li>Galkina, E., Ley, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte+influx+in+atherosclerosis.">Leukocyte influx in atherosclerosis.</a> Curr Drug Targets. 812, 1239-1248 (2007).</li><li>Bonanno, E., Mauriello, A., Partenzi, A., Anemona, L., Spagnoli, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+analysis+of+atherosclerotic+plaque+cells+from+human+carotids:+a+validation+study.">Flow cytometry analysis of atherosclerotic plaque cells from human carotids: a validation study.</a> Cytometry. 39, 158-165 (2000).</li><li>Liu-Wu, Y., Svenningsson, A., Stemme, S., Holm, J., Wiklund, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+analysis+of+macrophage-derived+foam+cells+from+human+atherosclerotic+lesions+by+using+a+"mock"+FL3+channel+in+flow+cytometry.">Identification and analysis of macrophage-derived foam cells from human atherosclerotic lesions by using a "mock" FL3 channel in flow cytometry.</a> Cytometry. 29, 155-164 (1997).</li><li>Galkina, E., Kadl, A., Sanders, J., Varughese, D., Sarembock, I. J., Ley, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphocyte+recruitment+into+the+aortic+wall+before+and+during+development+of+atherosclerosis+is+partially+L-selectin+dependent.">Lymphocyte recruitment into the aortic wall before and during development of atherosclerosis is partially L-selectin dependent.</a> J Exp Med. 203, 1273-1282 (2006).</li><li>Tung, J. W., Parks, D. R., Moore, W. A., Herzenberg, L. A., Herzenberg, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+approaches+to+fluorescence+compensation+and+visualization+of+FACS+data.">New approaches to fluorescence compensation and visualization of FACS data.</a> Clin Immunol. 110, 277-283 (2004).</li><li>Smith, E., Prasad, K. M., Butcher, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blockade+of+interleukin-17A+results+in+reduced+atherosclerosis+in+apolipoprotein+E-deficient+mice.">Blockade of interleukin-17A results in reduced atherosclerosis in apolipoprotein E-deficient mice.</a> Circulation. 121, 1746-1755 (2010).</li><li>Eid, R. E., Rao, D. A., Zhou, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-17+and+interferon-gamma+are+produced+concomitantly+by+human+coronary+artery-infiltrating+T+cells+and+act+synergistically+on+vascular+smooth+muscle+cells.">Interleukin-17 and interferon-gamma are produced concomitantly by human coronary artery-infiltrating T cells and act synergistically on vascular smooth muscle cells.</a> Circulation. 119, 1424-1432 (2009).</li></ol>

在这里，我们提出了一个基于流式细胞仪检测的免疫细胞组成的小鼠主动脉的调查方法。这种方法的主要优点是能够分析主动脉免疫细胞在单细胞水平和表征主动脉白细胞的激活状态。这种方法不仅限于小鼠主动脉和我们（未发表数据）和其他 10用这种方法来分析人体标本，如乳内动脉，主动脉瓣和冠状动脉。这种方法的一个限制是恢复单一主动脉小鼠主动脉白细胞数量相对较少。虽然?...

<table border="1"><tbody><tr><td> 材料 </td><td> 产品编号 </td><td> 公司 </td></tr><tr><td>胶原酶第十一</td><td> C7657 </td><td> Sigma - Aldrich公司</td></tr><tr><td>透明质酸</td><td> H3506 </td><td> Sigma - Aldrich公司</td></tr><tr><td>我脱氧核糖核酸酶，2型</td><td> D4527 </td><td> Sigma - Aldrich公司</td></tr><tr><td>胶原酶我</td><td> C0130 </td><td> Sigma - Aldrich公司</td></tr><tr><td>胶原酶二</td><td> LS004174 </td><td>沃辛顿生化公司</td></tr><tr><td>弹性蛋白酶</td><td> LS002292 </td><td>沃辛顿生化公司</td></tr></tbody></table>

flow cytometry analysis of immune cells within murine aortas

动脉粥样硬化是一种慢性炎症过程大中型船只的特点是形成泡沫细胞，免疫细胞，血管内皮细胞和平滑肌细胞，血小板，细胞外基质，具有广泛坏死富含脂质核心组成的斑块和周围组织纤维化1先天免疫和适应性免疫反应的武器是参与动脉粥样硬化的启动，发展和持久2，3是一个显着的证据机构，不同亚群的免疫细胞，如巨噬细胞，树突状细胞，T和B淋巴细胞，在目前的健康，易发生动脉粥样硬化小鼠 4主动脉。此外，免疫细胞被发现在周围，这表明这个组织的重要作用，在动脉粥样硬化的主动脉外膜2。 一段时间以来，不同类型的免疫细胞，它们的激活状态，和主动脉壁内细胞成分的定量检测RT - PCR和免疫组织化学方法对动脉粥样硬化的研究是有限的。作了一些尝试执行流式细胞仪利用人体的主动脉，和一些问题，如高自体荧光，已报告 5,6 。人类动脉粥样硬化斑块1胶原酶消化，收集游离细胞和CD14的染色+ /表面CD11c +突出的巨噬细胞源性泡沫细胞。在这项研究中，一个“模拟”通道使用，以避免假阳性染色。6坏死性材料，在消化过程中积累了大量的碎片，产生在主动脉样品的自体荧光上升。要解决这个问题，阴性和阳性对照小组已经提出，但只有双染色可以应用于这些样本。我们已经开发出一个新的流式细胞仪为基础的方法，分析免疫细胞的组成和特征的活化，增殖，免疫细胞分化的健康，易发生动脉粥样硬化主动脉。此方法使主动脉壁的免疫细胞组成的调查，并打开使用广谱的免疫学方法对这种疾病的免疫方面的调查的可能性。

Watch this Scientific Journal Video about Flow Cytometry Analysis of Immune Cells Within Murine Aortas at JoVE.com

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

本文介绍一个基于流式细胞仪的方法进行调查的主动脉免疫组成。该文件还说明了一个额外的技术，允许单独审视周围的外膜和血管壁。这种方法打开执行主动脉白细胞表型分析，适用于动脉粥样硬化研究的几种免疫检测的可能性。

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 ...

flow-cytometry-analysis-of-immune-cells-within-murine-aortas

Research

JoVE Journal

Immunology and Infection

34.3K 次观看.  Eastern Virginia Medical School. 本文介绍一个基于流式细胞仪的方法进行调查的主动脉免疫组成。该文件还说明了一个额外的技术，允许单独审视周围的外膜和血管壁。这种方法打开执行主动脉白细胞表型分析，适用于动脉粥样硬化研究的几种免疫检测的可能性。

视频: Flow Cytometry Analysis of Immune Cells Within Murine Aortas

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

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 ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

主要鼠Ⅱ型肺泡上皮细胞的功能及分子生物学研究的流式细胞仪分离

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

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 ...

Qualitative and Quantitative Analysis of the Immune Synapse in the Human System Using Imaging Flow Cytometry

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins towards the immune synapse to assure a stable binding and signal exchange. Classical confocal, TIRF, or super-resolution microscopy have been used to study the immune synapse. Since these methods require manual image acquisition and time-consuming quantification, the imaging of rare events is challenging. Here, we describe a workflow that enables the morphological analysis of tens of thousands of cells. Immune synapses are induced between primary human T cells in pan-leukocyte preparations and Staphylococcus aureus enterotoxin B (SEB)-loaded Raji cells as APCs. Image acquisition is performed with imaging flow cytometry, also called In-Flow microscopy, which combines features of a flow cytometer and a fluorescence microscope. A complete gating strategy for identifying T cell/APC couples and analyzing the immune synapses is provided. As this workflow allows the analysis of immune synapses in unpurified pan-leukocyte preparations and hence requires only a small volume of blood (i.e., 1 mL), it can be applied to samples from patients. Importantly, several samples can be prepared, measured, and analyzed in parallel.

Here, we describe a complete workflow for the qualitative and quantitative analysis of immune synapses between primary human T cells and antigen-presenting cells. The method is based on imaging flow cytometry, which allows the acquisition and evaluation of several thousand cell images within a relatively short period of time.

影像性流式细胞仪对人体免疫突触的定性和定量分析

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins ...

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. ...

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 ...