Name: isolation and flow cytometric analysis of immune cells from the ischemic mouse brain
Uploaded: 2016-02-12T15:00:00.000+00:00
Duration: 12 min 14 s
Description: Watch this Scientific Journal Video about Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain at JoVE.com

作者感谢伊莎贝尔·舒尔茨优秀的技术支持。

所有的动物实验必须符合动物保健根据标准 （例如，该指南实验动物卫生，NIH出版号85-23美国国立研究院公布的维护和使用的1996年修订），并有被批准由相应的国家权威机构。 1.试剂的制备<ol><li>消化缓冲液（1毫升每半球）:将纯化的消化酶，例如一个标准化的混合物，LIBERASE低嗜热菌浓度（TL）至2U / ml，在Hanks平衡盐溶液（HBSS）中含有的钙的浓度（Ca）和镁（Mg）的</li><li>洗涤缓冲液用DNA酶:将DNA酶I至在HBSS一个的666U / ml的浓度（钙/镁免费）含胎牛血清10％（FCS）的</li><li>含10％FCS的HBSS（钙/镁免费）:洗涤液无DNA酶</li><li>密度梯度介质（5毫升，每半球）:准备股票等渗密度梯度0;介质（SIP 100％）通过混合九个部分密度梯度介质的同一个部分的1.5M氯化钠。通过加入HBSS含FCS的3％的适当体积（钙/镁免费）稀释的SIP至25％的密度</li><li>流式细胞术（FC）缓冲液:含FCS的3％的准备的磷酸盐缓冲盐水（PBS） </li></ol> 2.短暂大脑中动脉闭塞注意:通过腔内缝合技术瞬态大脑中动脉闭塞（MCAO）是在12周龄雄性C57BL / 6小鼠18前面所述进行。 <ol><li>简言之，麻醉用100％氧气2.0％异氟烷的小鼠。维持体温在36.5℃下由一个反馈控制的加热装置±0.5℃。 </li><li>颈总动脉和颈外动脉结扎后，介绍在颈总动脉并推进它的标准化硅橡胶涂覆单丝对中间C的原点erebral动脉。 </li><li> 45分钟后，取出长丝，以允许再灌注。在假手术动物中，闭塞中脑动脉，以避免局部缺血之后立即撤回灯丝。 </li></ol> 3.穿心灌注和脑解剖<ol><li>由管的一端浸入冰冷的HBSS（钙/镁免费）制备蠕动灌注泵。固定钝23政针到灌注管的另一端和开关泵上以完全填充用HBSS（钙/镁免费）的管。 </li><li>深深地麻醉小鼠用4％的异氟醚和二氧化碳吸入安乐死。 </li><li>将鼠标背部嵌在一个塑料托盘的夹层板。传播脱颖而出并在尽可能广泛和解决这些问题与20G针的夹层板后爪。 </li><li>抓住腹部皮肤采用了直板1×2牙钳和用锋利的虹膜剪，使通过体壁和腹壁外侧切口和揭露肝。 </li><li>提起胸骨和用锋利/钝虹膜剪钝刀刃切割膜片。继续切割在两侧的横向肋笼caudocranial方向。要小心，不要伤害肺部，心脏和胸动脉。 </li><li>提起皮瓣用钝镊子，针它放在夹层板。使用钝头镊子和剪刀仔细的心脏结缔组织分开。 </li><li>保持心脏用钝头镊子和插入钝23政针与所连接的输液管的尖端插入左心室心尖。注意:不要太远针头插入左心室，避免室间隔伤害。如果有必要，修复到位套管用直锋利的钳。 </li><li>用锋利的虹膜剪切开右心房，并立即打开所述泵（流速:8 - 10ml /分钟）。 </li><li>继续灌注，直到肝显示出光咖色（〜30毫升的HBSS）。始终灌注，小心地避免在管道的任何气泡的形成。 </li><li>斩首直手术剪仅次于头颅鼠标。使用虹膜剪，使头皮正中切口，暴露头骨。 </li><li>将尖锐的虹膜剪一个尖端插入枕骨大孔和横向切入头骨。重复另一侧。 </li><li>用锋利的虹膜剪小心地从同一腔，迈向鼻子中线切割。尽量保持如过眼云烟尽可能剪刀年底，以避免大脑损伤。 </li><li>用细镊子轻轻地从每个大脑半球剥离颅骨。注意:梗塞组织可能会附着在脑膜或颅骨;确保不失去的脑组织由于不谨慎解剖</li><li>提起大脑用抹刀和用锋利的虹膜剪仔细剖析其固定在头骨颅神经纤维。放置脑入15毫升管填充用10ml HBSS（+钙/镁），并保持它那hortly冰上。 </li></ol> 4.解离脑组织的单细胞悬浮液<ol><li>小心地取出脑干及小脑用干净的刀片。使用干净的刀片到hemisect脑和切割沿冠状面每个半球成三件大致相等的尺寸。 </li><li>剁碎使用5毫升注射器的柱塞端解剖每个半球的组织通过100微米的细胞滤网。连续冲洗用冰冷的HBSS（+钙/镁）的细胞过滤网。在刨冰店匀浆样本。 </li><li>离心机在286 XG和4℃下5分钟，小心弃去上清液。重悬在1ml的消化缓冲液中的颗粒和悬浮液转移到2ml管中。在37℃下缓慢连续旋转孵育停牌1小时。 </li><li>通过一个70微米的细胞滤网筛细胞悬液，并用3毫升含DNA酶的洗涤缓冲液，接着15自由DNA酶洗涤缓冲液彻底冲洗。离心机在286 xg离心和18℃进行5分钟，并弃去上清液。注:注:使用DNA酶消除由细胞裂解可能导致细胞聚集损害细胞存活DNA粘液。 </li></ol> 5.密度梯度离心为髓鞘和细胞碎片的去除<ol><li>在5毫升25％的密度梯度介质和吸取的悬浮液至15毫升管悬浮细胞沉淀。通过反复轻柔吹打避免泡沫的形成调匀。注意:密度梯度介质应当在RT用于防止细胞聚集。 </li><li>离心机在521 xg离心和18℃20分钟。使用转子的最低加速曲线，让转子停不带刹车。轻轻地取出从离心机管无晃动。小心吸髓鞘涂层并将上清液，同时保留在管底部的细胞沉淀。注意:请务必删除整个髓鞘外衣任何剩余的将impaiR流量流式细胞仪分析。 </li><li>重悬在10毫升DNA酶游离洗涤缓冲液的颗粒和吸取悬浮液到一个新的15ml试管。注意:此洗涤步骤是关键的，因为充分除去密度梯度介质的显著有助于最终细胞样品的数量和质量。 </li><li>离心再次在286 xg离心和10℃5分钟并弃上清。重悬在100μl冷洗涤缓冲液中的细胞，并通过用血细胞计数器台盼蓝排除测定细胞计数和活力。在4℃储存的样本，迅速进一步处理它们。 </li></ol> 6.抗体染色的流式细胞仪分析<ol><li>之前抗体标记，孵育细胞悬浮液在4℃下用抗鼠CD16 / CD32的FC-受体阻断剂10分钟（2.5微克/毫升的最终浓度，稀释因子1:200），以防止非特异性结合。 </li><li>添加荧光团共轭的初级抗体在一个ppropriate浓度（如表 1所示），以细胞悬浮液，并在4℃下孵育在黑暗中20分钟。注:对照样品不与初级抗体染色，但其它处理相同。 </li><li>在350 xg离心洗涤细胞用2ml的FC缓冲器和离心机和10℃7 min.Carefully吸在200μlFC缓冲器的上清，重悬细胞沉淀。店内暂时的样品在4℃在黑暗中，直到流式细胞仪分析。 </li></ol> 7.绝对定量计数珠<ol><li>反向吸取FC缓冲器40微升和10微升细胞悬浮液到含有已知数量的荧光珠的计数管中。注:移液器校准提供准确的10微升样品作为白细胞计数的后续计量是指本卷注意。 </li><li>孵化用FITC标记的CD45抗体（最终浓度的细胞悬浮液的5微克/毫升，稀释因子1:100），在4℃在黑暗中20分钟。 </li><li>反向填补用200μl的FC缓冲器的计数管没有任何洗涤步骤，并直接记录的CD45 +细胞中的流式细胞仪。 </li></ol> 8.流式细胞仪采集注意:要分析所提出的抗体面板，适当的流式细胞仪已经被配备有蓝色（488纳米），红色（635纳米），紫（405纳米）的激光和过滤器的FITC，PE，PerCP经Cy5.5，PECy7， APC-Cy7的和AmCyan。 <ol><li>通过使用未染色细胞从局部缺血半球调整向前（FSC）和侧向（SSC）分散。区分从单个细胞双峰中示出的FSC的面积和高度的点图。 </li><li>显示在单参数柱状图所有的单细胞使用的每个荧光通道，调整光电倍增管（PMT）电压，使单个细胞显示在最左边的直方图。 </li><li>使用CD45-FITC单染色样品通过在每个直方图和散射图backgating CD45 高免疫细胞以检查感兴趣的细胞分散和PMT设置。注意:适应的FITC的PMT电压，使得CD45阴性，中间体（int）和高表达细胞能够彼此区别开来。 </li><li>用抗体捕获补偿珠进行多色彩补偿。设置闸门为小胶质细胞（CD45 int）和白细胞（CD45 高 ），并随后根据图 1中所显示的选通策略定义白细胞亚群。 </li></ol>

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Phenotypic differences defined and direct ex vivo antigen presentation to myelin basic protein-reactive CD4+ T cells compared.</a> Journal of immunology. 154 (9), 4309-4321 (1950).</li><li>Ajami, B., Bennett, J. L., Krieger, C., Tetzlaff, W., Rossi, F. a. b. i. o. M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+self-renewal+can+sustain+CNS+microglia+maintenance+and+function+throughout+adult+life.">Local self-renewal can sustain CNS microglia maintenance and function throughout adult life.</a> Nature neuroscience. 10 (12), 1538-1543 (2007).</li><li>Mildner, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+and+non-redundant+roles+of+microglia+and+myeloid+subsets+in+mouse+models+of+Alzheimer's+disease.">Distinct and non-redundant roles of microglia and myeloid subsets in mouse models of Alzheimer's disease.</a> The Journal of neuroscience. 31 (31), 11159-11171 (2011).</li><li>Ginhoux, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fate+mapping+analysis+reveals+that+adult+microglia+derive+from+primitive+macrophages.">Fate mapping analysis reveals that adult microglia derive from primitive macrophages.</a> Science. 330 (6005), 841-845 (2010).</li></ol>

作者宣称，他们没有竞争的经济利益。

在这里，我们描述了白细胞从鼠脑实验性卒中后的隔离的简单而有效的方法。该方法可靠地产生每大脑半球，允许在一个生物重复测量不同流板高度重复性细胞计数。 显然，不完全除去血，包括免疫细胞，将导致在该输入的缺血性脑炎症细胞的实际量的失真图。因此，使用该协议时，要特别注意彻底穿心灌注，以防止渗入免疫细胞与非炎症性循环白细胞的污染。从经验，对于左心室穿刺钝针的使用减少了对室间隔这将绕过循环的灌注损伤的风险。从鼻孔灌注流体出现 是，灌注pressureis过高，因而一个符号，一个空隙流速&gt; 10ml /分钟。以淡白色的脑组织表示良好的灌注，而粉红色的颜色是灌注不良的征兆。 此协议的另一个关键步骤是脑组织，它包括机械碎片以及酶消化高效分离。通过细胞过滤切碎组织是至关重要的，以提供改进的蛋白酶的效力。然而，均质的组织时，避免过大的压力，因为单核吞噬细胞是高度敏感的自溶22。用于酶促消化LIBERASE TL建议这是高度纯化的胶原酶I和II包含低浓度的中性蛋白酶嗜热菌的混合物。与由LIBERASE TL治疗（KM未发表的数据）显示活细胞显著更高恢复先前所述分离方案14,22,23的直接比较。相比胶原酶这是经常使用d表示从脑的免疫细胞的分离，LIBERASE TL含有内毒素的可忽略不计的水平。这是特别重要的，如果细胞被分类为下游分析，因为高含量的内毒素的可能会改变免疫细胞24的活化状态并严重损害细胞培养成果25。传统胶原酶的另一缺点 是在危害结果的可重复性，并要求每批26工作浓度的测定酶活性显著很多到很多不同之处。 在成人脑，髓鞘去除通过密度梯度离心是用于下游应用如流式细胞仪或对基因或蛋白质表达进一步的研究中不可缺少的步骤。该协议的一个主要优点是，它不需要梯度费时制备单层密度的过程。此外，分离协议产生可靠的结果由没有经验的实验者进行当s均匀。它不含接近彼此，很难吸移管的密度分层梯度之间没有干扰的接口。 根据表面标记物CD45的表达，协议产生的缺血性脑三大细胞类。绝大多数细胞属于那是由神经细胞，星形胶质细胞，室管膜细胞和内皮细胞CD45阴性人群。他们丰富的存在归因于单层密度梯度它的主要目的是有效的髓鞘和杂物清除。此外，代表驻地小胶质细胞27 CD45 INT人口可以从主要包含浸润血白细胞一个CD45 高人口加以区分。然而，应该指出的是，活化的小胶质可能采用血液出生骨髓细胞​​的表型和功能。因此，只有运用阅历ð技术，如联体28，骨髓嵌合体29或命运图谱分析30让炎症时这两个群体之间的明确区分。 在流式细胞术数据的分析往往局限于特定细胞群的百分比分布。然而，半球比较梗塞到非梗塞脑组织时此信息可能会产生误导，因为它不考虑这是由缺血性损伤改变总脑白细胞计数。因此，借鉴幅度和炎症浸润表型的全貌后，免疫细胞亚群的行程相对分布应绝对细胞数目的补充。当使用如在此协议中所述微珠，精确的样本体积的移液是绝对强制性获得可靠的结果。此外，强烈建议反向移液，以避免在计数管泡沫形成。所产生的气泡将减少微珠的预期体积并因此导致与样品中的绝对的CD45 高白细胞计数的高估。 总之，该协议提供了一种简单和可靠的方法来从脑分离的免疫细胞。它可以作为解剖到缺血性中风的炎症反应的复杂性的宝贵工具。未来的应用包括在传播和缺血性脑炎症的分辨率单核细胞亚群的依赖于时间的作用的调查。同样地，适应性免疫系统的行程后的作用是知之甚少。通过流量的原位特定子集转录因子或细胞因子的表达鉴定T细胞亚群的流式细胞仪分析可能有助于阐明其对中风后长期的恢复和新的治疗方法的发展从而开有希望的途径影响。

脑卒中触发的局部血流停止后迅速开始并涉及免疫系统的几乎所有部分的持续的炎症反应。这种炎症反应的一个重要特点是它是由脑血管内皮细胞，大量趋化因子分泌的活化驱动的，增加的交感神经流出1-3的免疫细胞向脑的定时涌入。炎症细胞浸润以前被认为是缺血性脑卒中有害的，但是，有几个治疗试验旨在阻止乱白细胞出口到大脑不能诱导可测量的临床益处4。最近，很明显，最初参与了缺血性脑损伤5进展单核细胞衍生的细胞也可能发挥关键作用的炎症和后续的组织修复6的分辨率。 由于表型和泛函的识别单核细胞和巨噬细胞之间Onal地区异质性，在发展和炎症的分辨率单核吞噬细胞的作用的知识已经显著扩大。在小鼠中，循环的单核细胞可以按照淋巴细胞抗原6复合物（LY-6C）7的在它们的表面表达被分类成至少两个功能不同的子集。而莱曼6C 高 &#39;inflammatory monocytes&#39;显然证明是细菌感染8的控制至关重要，其在无菌损伤作用更为争议。在缺血性中风，CCR2 +阮文黎-6C 高的单核细胞选择性消融导致出血性梗死改造6。然而，同样的实验方法脑出血后的9急性改善残疾。同样，T细胞的不同子集被认为在缺血性脑损伤发挥无论是有害的10或防护行动11，但是，数据controversi人12,13及认股权证进一步调查。鉴于这种复杂性不断增加，它变得清晰，对缺血损伤和修复的各种免疫细胞的作用更深入的知识对于翻译的实验结果为靶向治疗脑卒中后炎症的关键。 今天，分析细胞免疫应答的最有力的工具是多色流式细胞术。它允许在炎症部位 ​​的鉴定和各种免疫细胞亚群的定量而不需要偏压系统通过体内标记或遗传操作14。与抗体的细胞表面标记物的同时染色对细胞内细胞因子15或转录因子16另外提供关于个体，表型鉴定的细胞的功能的状态信息。作为一个主要的缺点，需要进行流式细胞分析单细胞悬浮液，从而在LOC信息细胞渗透的alization丢失。然而，虽然组织学是理想获得空间信息，它是由可在一个单一的时间被用于在特定的组织表征的免疫细胞亚群的抗体的数量的限制。今天，需要各种表面标记的存在和不存在的组合，炎症17中明确地识别罕见的免疫细胞亚群，尤其是不同的单核细胞衍生的细胞群体。 这里，我们描述了一种有效的协议，以高的数字白细胞从使用简单的单层密度梯度缺血后鼠脑分离。将得到的细胞悬浮液可以通过多维流中任一分析仪或进一步通过流式细胞仪分选或免疫磁性选择进行富集来执行高度特异性的下游分析。该方法详细介绍穿心灌注，清除大脑半球，脑组织的解离成单细胞停赛ensions，对髓鞘去除密度梯度离心以及抗体染色流式细胞仪分析。

<table><tbody><tr><td>Peri-Star Pro Peristaltic Pump, 4-channel</td><td>World precision instruments</td><td>PERIPRO-4LS</td><td></td></tr><tr><td>Heracell 240i CO2 incubator&amp;nbsp;</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>MACSmix Tube Rotator</td><td>Miltenyi Biotec GmbH</td><td></td><td></td></tr><tr><td>Heraeus Multifuge 3SR+</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>FACS Canto II</td><td>Becton Dickinson</td><td></td><td></td></tr><tr><td>Isoflurane</td><td>Abbott</td><td>4831850</td><td></td></tr><tr><td>Hank&#039;s buffered salt solution (HBSS)</td><td>Gibco/Life Technologies GmbH</td><td>14175-129</td><td></td></tr><tr><td>HBSS with Calcium/Magnesium</td><td>Gibco/Life Technologies GmbH</td><td>24020-091</td><td></td></tr><tr><td>Liberase TL</td><td>Roche Diagnostics GmbH</td><td>5401020001</td><td></td></tr><tr><td>Percoll Plus</td><td>GE Healthcare Europe GmbH</td><td>17-5445-01</td><td></td></tr><tr><td>Fetal bovine serum&amp;nbsp;</td><td>PAN Biotech</td><td>3302-P101003</td><td></td></tr><tr><td>Trypan blue</td><td>Gibco/Life Technologies GmbH</td><td>15250-061</td><td></td></tr><tr><td>Trucount</td><td>BD Biosciences</td><td>340334</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Biochrom AG</td><td>L 182-10</td><td></td></tr><tr><td>DNAse I</td><td>Roche Diagnostics GmbH</td><td>11284932001</td><td></td></tr><tr><td>CD11b Horizon V500</td><td>BD Biosciences</td><td>562128</td><td></td></tr><tr><td>CD16/CD32</td><td>eBioscience</td><td>14-0161</td><td></td></tr><tr><td>CD45.2 FITC</td><td>eBioscience</td><td>11-0454</td><td></td></tr><tr><td>CD3 PE</td><td>Biolegend</td><td>100307</td><td></td></tr><tr><td>CD19 PE-Cy7</td><td>Biolegend</td><td>115519</td><td></td></tr><tr><td>Ly6C APC-Cy7</td><td>BD Biosciences</td><td>560596</td><td></td></tr><tr><td>Ly6G PerCP-Cy5.5</td><td>BD Biosciences</td><td>560602</td><td></td></tr><tr><td>Silicone Tubing, 1 m</td><td>World precision instruments</td><td>503022</td><td></td></tr><tr><td>Fine Iris Scissors sharp</td><td>Fine Science Tools</td><td>14094-11</td><td></td></tr><tr><td>Surgical Scissors</td><td>Fine Science Tools</td><td>14130-17</td><td></td></tr><tr><td>Fine Iris Scissors sharp/blunt</td><td>Fine Science Tools</td><td>14028-10</td><td></td></tr><tr><td>Straight 1 x 2 teeth forceps</td><td>Fine Science Tools</td><td>11021-14</td><td></td></tr><tr><td>Blunt-end Forceps</td><td>Fine Science Tools</td><td>11008-13</td><td></td></tr><tr><td>5ml syringe plunger</td><td>Carl Roth GmbH (Braun)</td><td>EP96.1</td><td></td></tr><tr><td>Cell strainer, 100 &amp;micro;m</td><td>Dr. I. Schubert, BD</td><td>2360-00</td><td></td></tr><tr><td>Omnican Fine dosage syringe 1 ml</td><td>Braun</td><td>TBD</td><td></td></tr><tr><td>Cell strainer, 70 &amp;micro;m</td><td>Greiner Bio-One GmbH</td><td>542 070</td><td></td></tr><tr><td>FACS Tubes</td><td>BD Bioscience GmbH</td><td>352052</td><td></td></tr><tr><td>serological pipettes, 10 ml</td><td>Greiner Bio-One GmbH</td><td>607180</td><td></td></tr><tr><td>serological pipettes, 10 ml</td><td>Sarstedt AG&amp;amp;Co</td><td>861,254,025</td><td></td></tr><tr><td>serological pipettes, 25 ml</td><td>Greiner Bio-One GmbH</td><td>760180</td><td></td></tr><tr><td>serological pipettes, 5 ml</td><td>Greiner Bio-One GmbH</td><td>606180</td><td></td></tr><tr><td>serological pipettes, 25 ml</td><td>Sarstedt AG&amp;amp;Co</td><td>861,685,020</td><td></td></tr><tr><td>serological pipettes,5 ml</td><td>Sarstedt AG&amp;amp;Co</td><td>861,253,025</td><td></td></tr><tr><td>Tips, 0.1 - 10 &amp;micro;l</td><td>Corning B.V.Life Sciences</td><td>4840</td><td></td></tr><tr><td>Tips, 100 - 1,000 &amp;micro;l</td><td>Greiner Bio-One GmbH</td><td>740290</td><td></td></tr><tr><td>Tips, 10 - 200 &amp;micro;l</td><td>Greiner Bio-One GmbH</td><td>739296</td><td></td></tr><tr><td>Reaction tubes 1.5 ml</td><td>Greiner Bio-One GmbH</td><td>616201</td><td></td></tr><tr><td>Reaction tubes 2 ml</td><td>Greiner Bio-One GmbH</td><td>623201</td><td></td></tr><tr><td>Bacteriological petri dish, 94 x 16 mm</td><td>Greiner Bio-One GmbH</td><td>633180</td><td></td></tr><tr><td>Falcon 15 ml</td><td>Greiner Bio-One GmbH</td><td>188271</td><td></td></tr><tr><td>Falcon 50 ml</td><td>VWR International GmbH (BD)</td><td>734-0448</td><td></td></tr><tr><td>Neubauer hemocytometer</td><td>Biochrom AG</td><td>PDHC-N01</td><td></td></tr><tr><td>razor blade</td><td>Carl Roth GmbH</td><td>CK07.1</td><td></td></tr></tbody></table>

isolation and flow cytometric analysis of immune cells from the ischemic mouse brain

缺血性中风发起启动在血管内隔室，并涉及脑驻留细胞的快速激活一个健壮的炎症反应。这种炎症反应的关键机制是免疫细胞循环到缺血性脑由趋化因子释放和增加的内皮细胞粘附分子的表达促进的迁移。脑侵入白细胞是公知的有助于早期继发局部缺血损伤，但其对于炎症和以后脑修复的终止意义最近才被发现。 这里，提供了一种用于免疫细胞从缺血鼠脑的有效隔离的简单协议。穿心灌注后，大脑半球解剖和机械分离。用LIBERASE酶消化之后是密度梯度（如珀）离心去除髓鞘和细胞碎片。这个协议我的一个主要优点š不需要梯度费时制备，并且可以可靠地进行单层密度梯度程序。该方法产生每大脑半球高度重现细胞计数，并允许在一种生物复制测量几个流式细胞仪面板。实验中风后表型特征和脑入侵的白细胞数量可能有助于更好地理解缺血性损伤和修复其多方面的作用。

脑缺血经由左侧大脑中动脉（MCAO）的瞬态灯丝闭塞12周大的雄性C57BL / 6小鼠开始。对于假手术丝插入到阻塞左侧中脑动脉，并立即取出以便允许即时再灌注。重要的是，细胞神经炎症有很大不同常用的卒中模型19中。这具有外插从动物研究发现特别是当对人类中风的异质病理生理学要牢记。 小鼠处死24小时缺血后早期免疫细胞侵袭分析对缺血性脑。从半球缺血获得细胞计数（缺血同侧0.95±0.25×10E6）后，密度梯度离心法与那些从对侧大脑半球（缺血禁忌; 1.09±0.30点¯x10E6）和同侧假手术小鼠的半球（假IPSI; 1.12±0.18点¯x10E6，单向ANOVA，p值= 0.524）。通过台盼蓝排除测定的分离​​的细胞的存活率是高的，并没有在组（假96.85±0.60％，MCAO禁忌97.12±1.18％，MCAO IPSI 95.68±2.04％，单向ANOVA，P = 0.253之间显著不同）。 脑的组合物渗透24小时MCAO是通过流式细胞仪分析确定之后，图1示出了门控策略示意（A）和在一个代表性的笔划动物（B）。脑浸润白细胞被定义为CD45 高细胞，它可以从CD45 INT小胶质细胞分化。内CD45 高人口 ，中性粒细胞（PMN）由阮文黎-6G表达式标识，而T淋巴细胞被划定为高 CD45 CD3 +细胞。那么剩下的CD45 高细胞区分开由CD19（B淋巴细胞）和CD11b表达编。该细胞CD11b +部分进一步细分为阮文黎-6C 高 `炎症monocytes`和阮文黎-6C 低人口包括单核细胞，树突状细胞（DC）和巨噬细胞。 在中风急性期，骨髓免疫细胞主宰大脑渗透3,20。中性粒细胞血管闭塞后迅速进入大脑，促进炎症的单核细胞21外渗。 图2A显示LY6-G +中性粒细胞相比，对侧大脑半球和假手术缺血后缺血侧24小时增加的百分比。与此相反，CD3 + T细胞在缺血性半球的比例是（相对）比在健康脑低。内的CD11b +人口，脑缺血对移动阮文黎-6C 高炎症单核细胞强大的优势（图余额2B）。 除了 ​​相对分布，计数管被用来确定在CD45阳性事件的基础上的样品（图3）的免疫细胞亚群的绝对数。计数管含有冻干的颗粒中释放的已知数量的荧光珠。在样品中阳性细胞的绝对数量可以通过与细胞事件对珠粒事件来确定。计算每个半球使用以下等式CD45 高白细胞的数目:每个半球的CD45 高细胞=（CD45 高事件X总共计数珠/样品珠事件）×（总悬浮液体积（100微升）/试样体积（10微升））。 24小时MCAO后，在梗死半球的总白细胞计数显著相比增加对侧半球和假手术如（图3D，单向ANOVA，p值= 0.0004）。在迪计数<html> stinct免疫细胞亚群（PMN，T细胞，B细胞，LY-6C 高和LY-6C 低的单核细胞）可以容易地通过与各自的样品的总CD45 高的白血球数乘以它们的相对频率来计算。 <img alt="图1" src="/files/ftp_upload/53658/53658fig1.jpg" /> 图1.门控策略流式细胞术 （一）设门的示意图。 （B）显示大脑中动脉闭塞后，从有代表性的小鼠大脑24小时分离出白细胞的流式细胞仪分析。 FSC前向散射，中性粒细胞中性粒细胞，疾病预防控制中心的古典树突状细胞。 <a href="https://www.jove.com/files/ftp_upload/53658/53658fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> ftp_upload / 53658 / 53658fig2.jpg"/&gt; 图2.免疫细胞亚群的分化。阮文黎-6G + -neutrophils（A）的相对分布，CD3 + T细胞（A）和单核细胞亚群在同侧（IPSI）阮文黎-6C（B）的差异表达和对侧（标识禁忌）半球大脑中动脉闭塞（缺血）或相应的假手术后24小时。每个人口的百分比在栅极表示。 <a href="https://www.jove.com/files/ftp_upload/53658/53658fig2large.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/53658/53658fig3.jpg" /> 图3.脑白细胞的定量通过计数珠 。（A）示出了高度双阳性珠栅极。内的单细胞群（B）CD45 INT小胶质细胞可以从CD45 高白细胞（C）相鉴别。对于定量，门控CD45 高的事件的数量是归一化到计数珠事件。需要注意的是白细胞总数（CD45 高 ）计数显著同侧（IPSI）半球相比对侧（禁忌）半球假手术24大脑中动脉闭塞（MCAO）后的小时上升。 ** P &lt;0.01，*** P &lt;0.001通过单向ANOVA和Tukey`s事后多重比较检验。 N = 4 - 6每组。 FSC前向散射。 <a href="https://www.jove.com/files/ftp_upload/53658/53658fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table colgroup="" fo:keep-together.within-page="1"><tbody><tr><td rowspan="2"> 荧光 </td><td rowspan="2"> FITC </td><td rowspan="2"> PE </td><td> PerCP </td><td rowspan="2"> PC7 </td><td row跨度="2"&gt; APC-Cy7的</td><td rowspan="2"> V500 </td></tr><tr><td> （经Cy5.5） </td></tr><tr><td> 抗原 </td><td> CD45.2 </td><td> CD3 </td><td> LY-6G </td><td> CD19 </td><td> LY-6C </td><td>的CD11b </td></tr><tr><td> 最终浓度[微克/毫升] </td><td>五</td><td> 1 </td><td> 0.2 </td><td> 0.2 </td><td> 1 </td><td> 1 </td></tr><tr><td> 稀释因子 </td><td> 1/100 </td><td> 1/200 </td><td> 1 / 1,000 </td><td> 1 / 1,000 </td><td> 1/200 </td><td> 1/200 </td></tr><tr><td> 克隆 </td><td> 104 </td><td> 145-2C11 </td><td> 1A8 </td><td> 6D5 </td><td> AL-21 </td><td> M1 / 70 </td></tr></tbody></table> 表 1.基本 抗体混合物在缺血性脑免疫细胞识别。

Watch this Scientific Journal Video about Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain at JoVE.com

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

分离和流式细胞仪分析从缺血小鼠脑免疫细胞

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

isolation-flow-cytometric-analysis-immune-cells-from-ischemic-mouse

Research

JoVE Journal

Immunology and Infection

34.9K 次观看.  Fraunhofer Institute for Cell Therapy and Immunology. Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

视频: Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Isolation of Brain and Spinal Cord Mononuclear Cells Using Percoll Gradients

Isolation of immune cells that infiltrate the central nervous system (CNS) during infection, trauma, autoimmunity or neurodegeneration, is often required to define their phenotype and effector functions. Histochemical approaches are instrumental to determine the location of the infiltrating cells and to analyze the associated CNS pathology. However, in-situ histochemistry and immunofluorescent staining techniques are limited by the number of antibodies that can be used at a single time to characterize immune cell subtypes in a particular tissue. Therefore, histological approaches in conjunction with immune-phenotyping by flow cytometry are critical to fully characterize the composition of local CNS infiltration. This protocol is based on the separation of CNS cellular suspensions over discontinous percoll gradients. The current article describes a rapid protocol to efficiently isolate mononuclear cells from brain and spinal cord tissues that can be effectively utilized for identification of various immune cell populations in a single sample by flow cytometry.

The current article describes a rapid protocol to efficiently isolate mononuclear cells from brain and spinal cord tissues that can be effectively utilized for flow cytometric analyses.

大脑和脊髓的单核细胞的分离，使用Percoll梯度

Isolation of immune cells that infiltrate the central nervous system (CNS) during infection, trauma, autoimmunity or neurodegeneration, is often required ...

科研

免疫与感染

Stereological and Flow Cytometry Characterization of Leukocyte Subpopulations in Models of Transient or Permanent Cerebral Ischemia

Microglia activation, as well as extravasation of haematogenous macrophages and neutrophils, is believed to play a pivotal role in brain injury after stroke. These myeloid cell subpopulations can display different phenotypes and functions and need to be distinguished and characterized to study their regulation and contribution to tissue damage. This protocol provides two different methodologies for brain immune cell characterization: a precise stereological approach and a flow cytometric analysis. The stereological approach is based on the optical fractionator method, which calculates the total number of cells in an area of interest (infarcted brain) estimated by a systematic random sampling. The second characterization approach provides a simple way to isolate brain leukocyte suspensions and to characterize them by flow cytometry, allowing for the characterization of microglia, infiltrated monocytes and neutrophils of the ischemic tissue. In addition, it also details a cerebral ischemia model in mice that exclusively affects brain cortex, generating highly reproducible infarcts with a low rate of mortality, and the procedure for histological brain processing to characterize infarct volume by the Cavalieri method.

Brain myeloid cells characterization following stroke can be performed by stereology using the optical fractionator method, or by a flow cytometric analysis of brain leukocytes suspensions. Both are useful techniques to perform an accurate phenotypical distinction of the main myeloid cell subsets found in the ischemic brain.

白细胞亚群在暂时或永久的脑缺血模型体视学和流式细胞仪鉴定

Microglia activation, as well as extravasation of haematogenous macrophages and neutrophils, is believed to play a pivotal role in brain injury after ...

医学

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the &#946;-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of&#160;how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists&#8217; discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

分离和胶质瘤浸润外周血单个核细胞的流式细胞仪分析

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

Characterization of Immune Cells and Proinflammatory Mediators in the Pulmonary Environment

Immune cell expansion, activation, and trafficking to the lungs, which are controlled by the expression of multiple cytokines and chemokines, may be altered by severe brain injury. This is evidenced by the fact that pneumonia is a major cause of mortality in patients who have suffered from ischemic stroke. The goal of this protocol is to describe the use of multicolor flow cytometric analysis to identify 13 types of immune cells in the lungs of mice, including alveolar macrophages, interstitial macrophages, CD103+ or CD11b+ dendritic cells (DCs), plasmacytoid DCs, eosinophils, monocytes/monocyte-derived cells, neutrophils, lymphoid-derived&#160; T and B cells, NK cells, and NKT cells, following ischemic stroke induction by transient middle cerebral artery occlusion. Moreover, we describe the preparation of lung homogenates using a bead homogenization method, to determine the expression levels of 13 different cytokines or chemokines simultaneously by multiplex bead arrays coupled with flow cytometric analysis. This protocol can also be used to investigate the pulmonary immune response in other disease settings, such as infectious lung disease or allergic disease.

This protocol describes the use of flow cytometry to identify the changes in immune cell composition, cytokine profile, and chemokine profile in the pulmonary environment following transient middle cerebral artery occlusion, a murine model of ischemic stroke.

肺环境中免疫细胞和促炎介质的表征

Immune cell expansion, activation, and trafficking to the lungs, which are controlled by the expression of multiple cytokines and chemokines, may be ...

Flow Cytometry Approach to Characterize Phagocytic Properties of Acutely-Isolated Adult Microglia and Brain Macrophages In Vitro

Microglia and central nervous system (CNS)-infiltrating macrophages, collectively called CNS mononuclear phagocytes (CNS-MPs), play central roles in neurological diseases including neurodegeneration and stroke. CNS-MPs are involved in phagocytic clearance of pathological proteins, debris and neuronal synapses, each with distinct underlying molecular pathways. Characterizing these phagocytic properties can provide a functional readout that compliments molecular profiling of microglia using traditional flow cytometry, transcriptomics and proteomics approaches. Phagocytic profiling of microglia has relied on microscopic visualization and in vitro cultures of mouse neonatal microglia. The former approach suffers from limited sampling while the latter approach is inherently poorly reflective of the true in vivo state of adult CNS-MPs. This paper describes optimized protocols to phenotype phagocytic properties of acutely-isolated mouse CNS-MPs by flow cytometry. CNS-MPs are acutely isolated from adult mouse brain using mechanical dissociation followed by density gradient centrifugation, incubated with fluorescent microspheres or fluorescent A&#946; fibrils, washed, and then labeled with panels of antibodies against surface markers (CD11b, CD45). Using this approach, it is possible to compare phagocytic properties of brain-resident microglia with CNS-infiltrating macrophages and then assess the effect of aging and disease pathology on these phagocytic phenotypes. This rapid method also holds potential to functionally phenotype acutely-isolated human CNS-MPs from post-mortem or surgical brain specimens. Additionally, specific mechanisms of phagocytosis by CNS-MP subsets can be investigated by inhibiting select phagocytic pathways.

Assessment of phagocytic properties of microglia and brain macrophages can provide a valuable functional dimension to molecular profiling studies. We describe a validated protocol that uses flow cytometry to rapidly and reliably quantify phagocytosis of fluorescent microspheres and amyloid beta fibrils by acutely-isolated microglia and brain macrophages from mouse models.

Microglia and central nervous system (CNS)-infiltrating macrophages, collectively called CNS mononuclear phagocytes (CNS-MPs), play central roles in ...

神经科学

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 ...

从小鼠脑和颅骨骨髓中获得单细胞悬液

Isolating Immune Cells from Mouse Brain and Skull

Mounting evidence indicates that the immune response triggered by brain disorders (e.g., brain ischemia and autoimmune encephalomyelitis) occurs not only in the brain, but also in the skull. A key step toward analyzing changes in immune cell populations in both the brain and skull bone marrow after brain damage (e.g., stroke) is to obtain sufficient numbers of high-quality immune cells for downstream analyses. Here, two optimized protocols are provided for isolating immune cells from the brain and skull bone marrow. The advantages of both protocols are reflected in their simplicity, speed, and efficacy in yielding a large quantity of viable immune cells. These cells may be suitable for a range of downstream applications, such as cell sorting, flow cytometry, and transcriptomic analysis. To demonstrate the effectiveness of the protocols, immunophenotyping experiments were performed on stroke brains and normal brain skull bone marrow using flow cytometry analysis, and the results aligned with findings from published studies.

作者聚焦：研究脑缺血后的复杂免疫反应

To investigate the immune response to brain disorders, one common approach is to analyze changes in immune cells. Here, two simple and effective protocols are provided for isolating immune cells from murine brain tissue and skull bone marrow.

从小鼠大脑和颅骨中分离免疫细胞

Mounting evidence indicates that the immune response triggered by brain disorders (e.g., brain ischemia and autoimmune encephalomyelitis) occurs not only ...

分离和流式细胞仪分析从缺血小鼠脑免疫细胞

摘要

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此视频中的章节

大脑和脊髓的单核细胞的分离，使用Percoll梯度

白细胞亚群在暂时或永久的脑缺血模型体视学和流式细胞仪鉴定

分离和胶质瘤浸润外周血单个核细胞的流式细胞仪分析

肺环境中免疫细胞和促炎介质的表征

Flow Cytometry Approach to Characterize Phagocytic Properties of Acutely-Isolated Adult Microglia and Brain Macrophages In Vitro

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

从小鼠大脑和颅骨中分离免疫细胞

从小鼠大脑和颅骨中分离免疫细胞