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>

<ol>
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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 border="0" cellpadding="0" cellspacing="0" width="745">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Peri-Star Pro Peristaltic Pump, 4-channel</td>
			<td style="width:195px;">World precision instruments</td>
			<td style="width:119px;">PERIPRO-4LS</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heracell 240i CO2 incubator&#160;</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MACSmix Tube Rotator</td>
			<td>Miltenyi Biotec GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heraeus Multifuge 3SR+</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FACS Canto II</td>
			<td>Becton Dickinson</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isoflurane</td>
			<td>Abbott</td>
			<td align="right">4831850</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hank&#39;s buffered salt solution (HBSS)</td>
			<td>Gibco/Life Technologies GmbH</td>
			<td>14175-129</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HBSS with Calcium/Magnesium</td>
			<td>Gibco/Life Technologies GmbH</td>
			<td>24020-091</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Liberase TL</td>
			<td>Roche Diagnostics GmbH</td>
			<td>5401020001</td>
			<td>use at room temperature</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Percoll Plus</td>
			<td>GE Healthcare Europe GmbH</td>
			<td>17-5445-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine serum&#160;</td>
			<td>PAN Biotech</td>
			<td>3302-P101003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypan blue</td>
			<td>Gibco/Life Technologies GmbH</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trucount</td>
			<td>BD Biosciences</td>
			<td>340334</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate buffered saline</td>
			<td>Biochrom AG</td>
			<td>L 182-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNAse I</td>
			<td>Roche Diagnostics GmbH</td>
			<td>11284932001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD11b Horizon V500</td>
			<td>BD Biosciences</td>
			<td>562128</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD16/CD32</td>
			<td>eBioscience</td>
			<td>14-0161</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD45.2 FITC</td>
			<td>eBioscience</td>
			<td>11-0454</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD3 PE</td>
			<td>Biolegend</td>
			<td>100307</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD19 PE-Cy7</td>
			<td>Biolegend</td>
			<td>115519</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ly6C APC-Cy7</td>
			<td>BD Biosciences</td>
			<td>560596</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ly6G PerCP-Cy5.5</td>
			<td>BD Biosciences</td>
			<td align="right">560602</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Silicone Tubing, 1m</td>
			<td>World precision instruments</td>
			<td align="right">503022</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fine Iris Scissors sharp</td>
			<td>Fine Science Tools</td>
			<td>14094-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgical Scissors</td>
			<td>Fine Science Tools</td>
			<td>14130-17</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fine Iris Scissors sharp/blunt</td>
			<td>Fine Science Tools</td>
			<td>14028-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Straight 1x2 teeth forceps</td>
			<td>Fine Science Tools</td>
			<td>11021-14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blunt-end Forceps</td>
			<td>Fine Science Tools</td>
			<td>11008-13</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5ml syringe plunger</td>
			<td>Carl Roth GmbH (Braun)</td>
			<td>EP96.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell strainer, 100&#181;m</td>
			<td>Dr. I. Schubert, BD</td>
			<td>2360-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Omnican Fine dosage syringe 1ML</td>
			<td>Braun</td>
			<td>TBD</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell strainer, 70&#181;m</td>
			<td>Greiner Bio-One GmbH</td>
			<td>542 070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FACS Tubes</td>
			<td>BD Bioscience GmbH</td>
			<td>352052</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">serological pipettes, 10ml</td>
			<td>Greiner Bio-One GmbH</td>
			<td>607180</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">serological pipettes, 10ml</td>
			<td>Sarstedt AG&#38;Co</td>
			<td>861,254,025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">serological pipettes, 25ml</td>
			<td>Greiner Bio-One GmbH</td>
			<td>760180</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">serological pipettes, 5ml</td>
			<td>Greiner Bio-One GmbH</td>
			<td>606180</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">serological pipettes,25ml</td>
			<td>Sarstedt AG&#38;Co</td>
			<td>861,685,020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">serological pipettes,5ml</td>
			<td>Sarstedt AG&#38;Co</td>
			<td>861,253,025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tips, 0,1-10&#181;l</td>
			<td>Corning B.V.Life Sciences</td>
			<td>4840</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tips, 100-1000&#181;l</td>
			<td>Greiner Bio-One GmbH</td>
			<td>740290</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tips, 10-200&#181;l</td>
			<td>Greiner Bio-One GmbH</td>
			<td>739296</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reaction tubes 1,5ml</td>
			<td>Greiner Bio-One GmbH</td>
			<td>616201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reaction tubes 2ml</td>
			<td>Greiner Bio-One GmbH</td>
			<td>623201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bacteriological petri dish, 94x16mm</td>
			<td>Greiner Bio-One GmbH</td>
			<td>633180</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Falcon 15ml</td>
			<td>Greiner Bio-One GmbH</td>
			<td>188271</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Falcon 50ml</td>
			<td>VWR International GmbH (BD)</td>
			<td>734-0448</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Neubauer hemocytometer</td>
			<td>Biochrom AG</td>
			<td>PDHC-N01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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

34.8K 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

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

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

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

流式细胞仪分析小鼠主动脉内流的免疫细胞

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

科研

免疫与感染

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.

Bone marrow transplantation provides a way to change the genotype of the bone marrow derived cells. If the gene of interest is expressed in both bone marrow derived cells and non-bone marrow derived cells, bone marrow transplantation can change the bone marrow derived cells to a different genotype without changing the non-bone marrow derived cell genotype.

区分功能基因表达的作用，通过骨髓移植免疫和非免疫细胞在小鼠结肠炎

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice ...

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 ...

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

We describe a method for isolation and characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. Infection of susceptible mouse-strains with this parasite strain results in the induction of experimental cerebral malaria, a neurologic syndrome that recapitulates certain important aspects of Plasmodium falciparum-mediated severe malaria in humans 1,2 . Mature forms of blood-stage malaria express parasitic proteins on the surface of the infected erythrocyte, which allows them to bind to vascular endothelial cells. This process induces obstructions in blood flow, resulting in hypoxia and haemorrhages 3 and also stimulates the recruitment of inflammatory leukocytes to the site of parasite sequestration.
Unlike other infections, i.e neutrotopic viruses4-6, both malaria-parasitized red blood cells (pRBC) as well as associated inflammatory leukocytes remain sequestered within blood vessels rather than infiltrating the brain parenchyma. Thus to avoid contamination of sequestered leukocytes with non-inflammatory circulating cells, extensive intracardial perfusion of infected-mice prior to organ extraction and tissue processing is required in this procedure to remove the blood compartment. After perfusion, brains are harvested and dissected in small pieces. The tissue structure is further disrupted by enzymatic treatment with Collagenase D and DNAse I. The resulting brain homogenate is then centrifuged on a Percoll gradient that allows separation of brain-sequestered leukocytes (BSL) from myelin and other tissue debris. Isolated cells are then washed, counted using a hemocytometer and stained with fluorescent antibodies for subsequent analysis by flow cytometry.
This procedure allows comprehensive phenotypic characterization of inflammatory leukocytes migrating to the brain in response to various stimuli, including stroke as well as viral or parasitic infections. The method also provides a useful tool for assessment of novel anti-inflammatory treatments in pre-clinical animal models.

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

A method for isolation of adherent inflammatory leukocytes from brain blood vessels of Plasmodium berghei ANKA-infected mice is described. The method allows quantification as well as phenotypic characterization of isolated leukocytes after staining with fluorescent antibodies and subsequent analysis by flow cytometry.

脑幽静的白细胞的分离与分析<em&gt;伯氏疟原虫</emANKA感染的小鼠

We describe a method for isolation and  characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. ...

Isolation of Adipose Tissue Immune Cells

The discovery of increased macrophage infiltration in the adipose tissue (AT) of obese rodents and humans has led to an intensification of interest in immune cell contribution to local and systemic insulin resistance. Isolation and quantification of different immune cell populations in lean and obese AT is now a commonly utilized technique in immunometabolism laboratories; yet extreme care must be taken both in stromal vascular cell isolation and in the flow cytometry analysis so that the data obtained is reliable and interpretable. In this video we demonstrate how to mince, digest, and isolate the immune cell-enriched stromal vascular fraction. Subsequently, we show how to antibody label macrophages and T lymphocytes and how to properly gate on them in flow cytometry experiments. Representative flow cytometry plots from low fat-fed lean and high fat-fed obese mice are provided. A critical element of this analysis is the use of antibodies that do not fluoresce in channels where AT macrophages are naturally autofluorescent, as well as the use of proper compensation controls.

Adipose tissue (AT) is a site of intense immune cell activation and interaction. Almost all cells of the immune system are present in AT and their ratios are altered by obesity. Proper isolation, quantification, and characterization of AT immune cell populations are critical for understanding their role in immunometabolic disease.

脂肪组织免疫细胞的分离

The discovery of increased macrophage infiltration in  the adipose tissue (AT) of obese rodents and humans has led to an  intensification of interest in ...

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 ...

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both microbial and dietary. Given an increasing understanding that these luminal antigens help shape the immune response and that education of immune cells within the intestine is critical for a number of systemic diseases, there has been increased interest in characterizing the intestinal immune system. However, many published protocols are arduous and time-consuming. We present here a simplified protocol for the isolation of lymphocytes from the small-intestinal lamina propria, intraepithelial layer, and Peyer&#39;s patches that is rapid, reproducible, and does not require laborious Percoll gradients. Although the protocol focuses on the small intestine, it is also suitable for analysis of the colon. Moreover, we highlight some aspects that may need additional optimization depending on the specific scientific question. This approach results in the isolation of large numbers of viable lymphocytes that can subsequently be used for flow cytometric analysis or alternate means of characterization.

There is growing interest in the quantitative characterization of intestinal lymphocytes owing to increasing recognition that these cells play a critical role in a variety of intestinal and systemic diseases. In this protocol, we describe how to isolate single cell populations from different small-intestinal compartments for subsequent flow cytometric characterization.

小鼠小肠淋巴细胞的分离和流式细胞表征

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both ...

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 ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 &#181;m in diameter in inhalable particulate matter (PM10, particle size &#8804;10 &#181;m in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions.
So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles &#62;0.5 &#181;m.

Here, we present a protocol to quantify allergen-loaded particles by flow cytometry. Ambient particulate matter particles may act as carriers of adsorbed allergens. We show here that flow cytometry, a method widely used to characterize suspended solids &#62;0.5 &#181;m in diameter, can be used to measure these allergen-loaded particles.

粒子结合Bet v 1的变应原PM10的流式细胞分析

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore essential for understanding pathogenicity of different pathogens including HIV. Gamma delta (GD) T cells are the prototype of &#39;unconventional&#39; T cells and represent a relatively small subset of T cells defined by their expression of heterodimeric T-cell receptors (TCRs) composed of gamma and delta chains. This sets them apart from the classical and much better known CD4+ helper T cells and CD8+ cytotoxic T cells that are defined by alpha-beta TCRs. GD T cells often show tissue-specific localization and are enriched in epithelium. GD T cells orchestrate immune responses in inflammation, tumor surveillance, infectious disease, and autoimmunity.
Here, we present a method to reproducibly isolate and analyze human endocervical intraepithelial GD T lymphocytes. We have used endocervical cytobrush samples from women participating in the Women&#39;s Interagency HIV Infection Study (WIHS). Knowledge about GD T cells interactions during conditions in which there is an insult to the vaginal mucosal could be applied to any clinical study in which mucosal vulnerability is addressed, including the development of vaginal microbicides.In addition, knowledge about mucosal GD T cell responses has potential for application of GD T cell-based immune therapy in treating infectious diseases.

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

分离和流式细胞仪分析从缺血小鼠脑免疫细胞

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