这项工作是由美国心脏协会奖学金奖（16POST29950007）到FL，从健康（NIH T32 HL069765），以BLD，美国心脏协会奖学金奖（14POST20420025）至MA萨利赫全国学院培训津贴，以及美国国立卫生研究院支持K08奖（HL121671），男男性接触者。 MSM也由吉利德科学公司研究基金支持

范德比尔特大学的机构动物护理和使用委员会已批准本文所述的程序。小鼠被收容并按照指南实验动物的护理和使用照顾（美国国家科学院出版社2010年修订）。 1.主动脉淋巴结，肾和主动脉的小鼠隔离<ol><li>安乐死的CO 2吸入老鼠。喷用70％乙醇的胸部和小心地打开皮肤和胸壁用剪刀以暴露心脏。 </li><li>灌注脉管，在右心房执行一个小切口，并稳定地注入至少10毫升冷的PBS（约1毫升/秒）的成使用21或23政针左心室的心尖。灌注，直到所有机关烫一下。的心脏第一次Blanches的。肝脏热烫表明灌注已经很好完成。 </li><li>用小镊子和剪刀精，轻轻切开，拉出肠子，胃，脾，胰腺和肝脏，以更好地可视化的主动脉。 注意:此步骤必须非常精确地完成作为破坏胃肠道可诱发污染。 </li><li>切出来，删除每个肺用剪刀。冲洗使用无针注射器用磷酸缓冲盐水（PBS）的胸腔。用无菌纱布去除多余的血液和液体。 </li><li>取出用细镊子解剖腹主动脉淋巴结。确保不破裂胶囊。与解剖镊每个淋巴结的表面上除去剩余的脂肪。切出两肾细剪刀。 </li><li>解剖整个主动脉（胸，腹主动脉）有细，剪子弯。开始在心脏的电平，并仔细解剖从食道和椎骨向下至髂分叉确保保持附着到主动脉周围血管周围脂肪的水平距离。 注:主动脉进一步解剖以圣雷莫已经周围结构可以在显微镜下进行。具体而言，除去动脉分支（颈动脉，锁骨下动脉，腹腔干，肠系膜和肾动脉）和淋巴结。别保持的左右各主动脉血管周围脂肪一致层，因为这是在许多炎性细胞驻留在高血压的设置的部位。将含冷PBS分离的管各组织。 </li></ol> 2.从生成每个组织单细胞悬浮液<ol><li> 肾 注:肾脏可以通过结合机械解离加酶消化来解离成单细胞悬浮液。 <ol><li>通过加入胶原酶D（2毫克/毫升）和DNA酶I（100微克/毫升），以RPMI1640培养基（补充有5％胎牛血清（FBS））制备肾消化溶液。每个准备样品肾脏10毫升</li><li>使用镊子将两个肾脏转移到装有10ml肾消化解离管离子溶液。 </li><li>执行根据制造商的说明使用半自动化的离解器装置中的机械解离。 </li><li>机械解离后，分离从设备管并执行酶促消化。孵育样品在37ºC下连续旋转20分钟。通过添加补充有5％FBS的冷的RPMI 1640培养基立即停止酶促消化。 </li><li>通过移液将溶液转移到50毫升锥形管通过40微米的过滤器过滤之后。通过用1ml注射器的活塞加压分开逗剩余组织。沉淀细胞（500 XG，8分钟，4℃）。 </li><li>重悬沉淀于3ml 36％的密度梯度离心介质。转移悬浮液到15毫升锥形管中。轻轻加3毫升72％的密度梯度离心介质的3ml的36％的细胞悬浮液的下方而没有任何中断。离心机（1000 XG不带刹车，20分钟，4℃）。&lt;/ LI&gt; <li>免疫细胞将位于界面。删除含有细胞碎片最上面的黄色的层，然后把音量使用冷PBS15毫升。摇匀击穿梯度。旋细胞（300 XG，8分钟，4℃），重悬沉淀在3毫升的RPMI有5％FBS。指望使用台盼蓝排除血球活细胞的数量。 </li></ol></li><li> 主动脉 <ol><li>通过加入胶原酶A（1毫克/毫升），胶原酶B（1毫克/毫升）制备主动脉消化溶液中，DNA酶I（100微克/毫升），以RPMI1640培养基（补充有5％FBS）中。 </li><li>用细镊子，转移到主动脉含1毫升主动脉消解液2毫升管。切碎整个主动脉成小片，在1ml的消解液精剪。置于冰上。孵育样品为下连续旋转，在37℃30分钟。 </li><li>该解决方案转移到组织培养皿和停止enzyma通过加入冷的RPMI 5倍体积有5％FBS抽动消化。 </li><li>通过移液将溶液转移到50毫升锥形管通过40微米的过滤器过滤之后。通过用1ml注射器的活塞加压分开逗剩余组织成一个单一的细胞悬浮液。冲洗过滤器和颗粒细胞（300 XG，8分钟，4℃）。 </li><li>通过加入10ml的RPMI有5％FBS洗涤沉淀。旋细胞（300 XG，8分钟，4℃），重悬沉淀在3毫升的RPMI有5％FBS。指望使用台盼蓝排除血球活细胞的数量。 </li></ol></li><li> 主动脉淋巴结 <ol><li>放置一个40μm的过滤器在组织培养皿（60毫米×15毫米）。放置在过滤器的淋巴结和通过用1ml注射器的活塞加压分开逗他们成单细胞悬浮液。冲洗用2ml的RPMI有5％FBS的过滤器。收集在盘中的细胞。 </li><li>转让将细胞入15ml falcon管和沉淀细胞（300 XG，8分钟，4℃）。重悬在500微升的RPMI有5％FBS的颗粒。指望使用台盼蓝排除血球活细胞的数量。 </li></ol></li></ol> 3. 离体刺激细胞因子流式细胞仪检测<ol><li>制备细胞培养基:RPMI有5％FBS，非必需氨基酸为0.1mM，50μMβ巯基乙醇和1％青霉素/链霉素。 </li><li>离心机从肾脏，主动脉和淋巴结获得的每个细胞悬液（300 XG，8分钟，4℃）。以每毫升1×10 6个细胞的浓度重悬在细胞培养基每个粒料。 </li><li>通过加入2微升细胞活化鸡尾酒（含有佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA），钙离子载体离子霉素，和高尔基抑制剂布雷菲德菌素A）的每1 ml细胞培养物刺激细胞。 注:终浓度各组分的s为:的PMA 81纳米，用PMA 1.33μM离子霉素和5微克/毫升布雷菲德菌素A.治疗和离子霉素激活细胞产生的细胞因子。细胞因子分泌，必须截留在细胞中检测到的蛋白质。布雷菲德菌素一个使正常分泌的蛋白质的内质网和高尔基体的积累。因此，这里所描述的技术可增强细胞因子产生的淋巴细胞通过流式细胞术可检测性。 </li><li>板1〜2百万细胞在12孔（主动脉或肾）或24孔（淋巴结）的低粘附板。放置板在37℃增湿的 CO 2培养箱中培养3小时。 注:铺板的细胞的数目可以是在淋巴结下从对照小鼠。此外，刺激的时间必须为每个细胞群和细胞因子研究根据经验确定。在从主动脉，肾或淋巴结中分离的T细胞的情况下，我们建议刺激3〜4小时。较长的刺激W¯¯生病不增加细胞因子的分泌，并会诱发多种T细胞表面标志CD3一样，CD4和CD8进一步下调。 （请注意，即使3-4小时的刺激会诱发这些标记的一些下调。） </li></ol> 4.表面染色<ol><li>转移细胞进入聚苯乙烯FACS管和离心机（300 XG，8分钟，4℃）。用FACS缓冲液洗涤细胞两次（ 钙和镁+含4％FBS和2mM的EDTA的自由PBS中），并等分细胞悬浮液成根据所需抗体的面板的数目和所需的控制管的数目不同FACS管（见下文）。 </li><li>要执行每个小组的补偿，未染色准备对照管和单管染色每个组织类型和抗体的面板。此外，通过将所有，但抗体之一制备用于各抗体面板荧光减一（FMO）对照管。 </li><li>调整各管的体积至2ml用FACS缓冲。离心细胞悬浮液（300 XG，8分钟，4℃），通过孵育细胞与抗CD16 / CD32抗体的0.5微克除去上清液和块Fc受体每百万个细胞，在冰上10分钟。 注:CD16是低亲和力的IgG Fc受体III（FCR III）和CD32是Fc受体II。 CD16 / CD32是在粒细胞，肥大细胞，单核细胞/巨噬细胞，自然杀伤细胞，B细胞，树突状细胞和某些活化成熟T细胞中表达。 </li><li>执行生存力染色:在1000微升1×PBS中的用1×PBS，离心（300 XG，8分钟，4℃），重悬洗涤细胞。加入1微升细胞透性胺活性染料（染色的可行性）的。孵育在4℃下15分钟，避光。 </li><li>在温育后，在FACS缓冲液的适当体积制备抗体（用于表面染色）的鸡尾酒。用1-2毫升FACS缓冲液洗涤细胞两次，离心（300 XG，8分钟，4℃）并重新悬浮用100μl每个粒料的抗体混合物。孵育在4℃下30分钟，避光。 注:对于每一个流式细胞术抗体，它是有用的，以确定抗体的通过进行剂量滴定曲线所需的最佳量。 </li><li>用2ml FACS缓冲液洗涤细胞两次，并沉淀细胞（300 XG，8分钟，4℃）。 </li></ol> 5.固定，通透性和细胞内染色<ol><li>执行与通过按照制造商的说明，含有合适的缓冲液的固定和透化试剂盒中的细胞内染色。固定并通过在适当的缓冲液中重悬浮这些透化细胞。孵育的样品在4℃下40分钟，避光。 </li><li>加入1 ml 1X通透性和洗直接缓冲到固定和通透的细胞。离心机（500 XG，8分钟，4℃），去除上清。悬浮颗粒用2毫升1X洗涤缓冲液。 </li><li>制备antib的混合物odies（仅用于细胞内染色）在1×洗涤缓冲液（每管通常为100微升）的一个适当的量。 注:如上所述，最佳抗体浓度需要在面板中的每个抗体来确定。例如，对于抗体与IL-17A或IL-17F，我们使用的1:30的稀释因子，但对于抗体IFNγ和T-bet的，我们使用的1稀释因子:100。 </li><li>离心将细胞（500 XG，8分钟，4℃）并重新悬浮用100μl的抗体混合物的各粒料。孵育在4℃下40分钟，避光。 </li><li>用2ml 1×洗涤缓冲液洗涤细胞两次，并沉淀细胞（500 XG，8分钟，4℃）。悬浮颗粒用300μl1X洗涤缓冲和流量与适当的过滤器分析仪。 </li><li>对于每个组织样品的细胞的总数的定量，使用计数珠。之前获取，添加计数珠每个管的推荐的体积/数。 </li></ol> 6.补偿，门控，规范化和技巧<ol><li> 补偿和浇口 <ol><li>补偿运行未染色和单染色控制管，以调整光谱重叠。 注意:使用的补偿珠而不是基于细胞的补偿控制是必要的情况下，样品中的兴趣或细胞群的抗原存在在如此低的水平，使用的细胞将是困难的补偿。还值得注意的，使用串联染料时，最好因为串联染料变化很大到大量和与每个天以制备用于每个实验补偿。 </li><li>开始一个新的多色实验运行时，荧光减去面板所有的荧光团人控制（FMOs）。 注:FMO控件是包含所有在面板除了一个抗体的样品。这些控件允许阳性与阴性信号进行精确判别为省略抗体正常adjust为门。这些控制时的表达水平低，或者当目标人群的分离是不良（例如，当目标是细胞因子或转录因子）特别重要。虽然并不总是必要的，在某些情况下，同种型对照可代替优选的FMO控制，因为它们提供了抗体同种型和荧光团共轭无抗原的特异性适当的补偿。 </li><li>设置主门:调整前向散射面积（FSC-A）和侧向散射面积（SSC-A）的电压，以检测白细胞人口。 </li><li>排除死细胞。 注:活力染色与存在于表面和细胞内部上都游离胺反应。在对比活细胞，死细胞（受损的膜）允许染料进入细胞质，增加蛋白标记的量。因此，死细胞会比活细胞可方便的歧视亮。门上的活细胞。 注意:从主动脉和肾细胞悬浮液的存活率可能比从淋巴结细胞悬浮液的生存能力降低由于额外的消化步骤。 </li><li>绘图前向散射面积（FSC-A）[Y轴]和前向散射高度（FSC-H）x轴。汗衫出现在这个点图对角线。门上的这个角排除双峰。然后绘制SSC-A [Y轴]和CD45 [x轴。 </li><li>该CD45 +白细胞群容易识别。门上的这部分人口。随后群可以从这个人口被识别。 </li></ol></li><li> 细胞计数正常化 <ol><li>算珠具有导致低FSC和高的SSC特性。例如，如果50000珠加入到300微升的单细胞悬浮液中，出于任何绝对数量的计算公式得到的细胞类型每管如下: 细胞数p器管=（由流动计数仪珠50,000 /数）计数的细胞的x个。 利用该计算，能够确定每管任何给定细胞类型的绝对数量。根据多少管从每个器官生成，可以计算每个器官的细胞的数目。 注意:保持所有细胞和试剂在4℃的协议中。避免剧烈涡旋，因为它可以破坏细胞。使用最少的力量以沉淀细胞。避免在FACS管，因为它可以沉淀的细胞死亡使得泡沫。不要让小球在任何时候干出来的。 </li></ol></li></ol>

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K., Roederer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytometry:+Today's+technology+and+tomorrow's+horizons.">Cytometry: Today's technology and tomorrow's horizons.</a> Methods. 57 (3), 251-258 (2012).</li><li>Jiang, L., Tixeira, R., Caruso, S., 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=Monitoring+the+progression+of+cell+death+and+the+disassembly+of+dying+cells+by+flow+cytometry.">Monitoring the progression of cell death and the disassembly of dying cells by flow cytometry.</a> Nat Protoc. 11 (4), 655-663 (2016).</li><li>Lemoine, S., Jaron, B., Tabka, S., 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=Dectin-1+activation+unlocks+IL12A+expression+and+reveals+the+TH1+potency+of+neonatal+dendritic+cells.">Dectin-1 activation unlocks IL12A expression and reveals the TH1 potency of neonatal dendritic cells.</a> J Allergy Clin Immunol. 136 (5), 1355-1368 (2015).</li><li>Gaublomme, J. T., Yosef, N., Lee, Y., 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=Single-Cell+Genomics+Unveils+Critical+Regulators+of+Th17+Cell+Pathogenicity.">Single-Cell Genomics Unveils Critical Regulators of Th17 Cell Pathogenicity.</a> Cell. 163 (6), 1400-1412 (2015).</li></ol>

MSM是由吉利德心血管科学的资助。

The protocol described herein has been optimized to properly identify T cell subsets present within murine kidneys, aorta and lymph nodes. This protocol can be easily adapted to examine other immune cell subsets such as B lymphocytes and innate immune cells and can be modified to include other tissue types. The digestion step is critical and has to be modified and optimized for each tissue9. A prolonged digestion step or the use of an inappropriate enzyme can affect the stability of antigen expression. Similarly, an incomplete digestion can affect the results leading to a lower number of positive cells. Furthermore, the time of ex vivo stimulation needs to be carefully adjusted for each cell type/cytokine to allow for optimal stimulation with minimal cell toxicity. 
Although intracellular staining coupled with flow cytometry analysis is one of the most powerful methods to analyze immune processes at a cellular level in blood and tissues, this technique has its limitations. These limitations include the relatively low number of immune cells in cardiovascular and renal tissues. Thus, depending on the cell type examined, it may be necessary to pool organs from several mice to obtain one sample. Furthermore, the instability of markers is another limitation as the expression of markers can be affected by several steps during the process. For this reason, it is advisable to perform histology in parallel to confirm the results obtained by flow cytometry. There are also special considerations regarding the use of the flow cytometer and the interpretation of the results. The flow cytometer is a sophisticated instrument and requires highly trained operators. Importantly, despite the use of compensation controls, there is still an issue of spectral overlap that limits experiments to 8-12 markers (usually 8) per sample. 
Moreover, this technique is very sensitive and subject to several experimental manipulations which can lead to slight day-to-day variation in the quantification of results. Thus, if performing this technique across multiple days for a given study, it is important to include control and experimental groups each day to determine relative changes. Finally, another limitation/source of variation is the choice of the markers used to identify a population10. Each study tends to use different combinations of markers and fluorochromes to define a population, which can lead to slight differences in quantification.
Despite these limitations, this is a powerful experimental technique that allows the identification and quantification of T cell subsets in solid organs. In fact, while other techniques measure either the expression of surface markers, cytokines or transcription factors separately, this method permits the measurement of several markers simultaneously at the single cell level. Furthermore, once the cells are identified, several other parameters can then be determined11,12,13,14. For example, one can evaluate for markers of apoptosis, protein phosphorylation or the proliferation of a specific T cell subset15. In addition, there are newer proprietary methods to detect mRNAs in cells using flow cytometry16,17.
In conclusion, we have outlined a detailed and reproducible protocol to identify T cell subsets using surface and intracellular markers (cytokines and transcription factors) in murine kidney, aorta and aortic draining lymph nodes in a model of angiotensin II induced hypertension. This protocol can be applied to other tissues (with slight modification) and to other models of disease.

最近的证据表明，适应性免疫系统，特别是T淋巴细胞，在几个心血管疾病1,2,3,4,5的发展中发挥了关键作用。例如，在血管紧张肽II引起的高血压模型中，T细胞在血管和小鼠的肾脏的累积已经描述6。血管积累主要是在外膜和血管周围的脂肪。在肾脏中，T细胞堆积在髓质和肾皮质两者。取决于哪个子集参与，这些T细胞产生不同的细胞因子，可以影响血管和肾功能，并导致病理的发展（由麦克马斯特等 6中综述）。 CD4 + T辅助淋巴细胞可分为几个子集:根据其职能和签名CYTO T辅助1（Th1细胞），Th2细胞，TH9，Th17细胞，TH22，调节性T（Treg细胞）细胞和T滤泡辅助（Tfh发挥细胞）基恩斯7。同样，CD8 +细胞毒性T细胞可以被分类为TC1，TC2，TC17或TC9 8。也有（不表达的CD4或CD8 T细胞的标志物即细胞）双阴性T细胞。这些细胞的一个子集具有一个备用的γδT细胞受体（而不是经典α和β受体），因此，被称为的γδT细胞。通过表面标记，细胞因子和转录因子的流式细胞仪的多参数分析构成，以确定这些细胞中的最好的方法。虽然这种方法是在免疫学领域广泛使用，这是不那么在实体器官和在心血管疾病的设置说明。 从历史上看，在组织中的淋巴细胞的识别仅限于免疫组化或RT-PCR的方法。虽然免疫组织化学和免疫功能强大的方法来确定一个int抗原的组织分布erest，他们是不足以表型鉴定所涉及的子集。此外，虽然RT-PCR分析是检测抗原，细胞因子或转录因子的mRNA表达是有用的，它不允许在单个细胞水平同时多种蛋白质的检测。 流的出现术，特别是当与细胞内染色组合以检测细胞因子和转录因子，提供了强大的技术，其允许鉴定和定量在固体器官的免疫细胞亚群的单个细胞水平的调查。我们已经优化了细胞内染色法，流式细胞仪鼠肾，主动脉和主动脉淋巴结内出现在血管紧张素II诱发的高血压模型中的主要T细胞亚群鉴别。在一个高度重新组织消化， 离体活化，透化，并且表面和细胞内染色结果:每一步的优化可生产测定可应用于其他心血管和肾脏疾病模型。

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			<td height="21" style="height:21px;width:381px;">Collagenase D</td>
			<td style="width:257px;">ROCHE</td>
			<td style="width:177px;">11088882001</td>
			<td style="width:259px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Collagenase A</td>
			<td>ROCHE</td>
			<td>10103586001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase B</td>
			<td>ROCHE</td>
			<td>11088815001</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Dnase</td>
			<td>ROCHE</td>
			<td>10104159001</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">1X Red blood cell lysis buffer</td>
			<td>eBioscience</td>
			<td>00-4333-57</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">RPMI Medium 1614 1X</td>
			<td>Gibco</td>
			<td>11835-030</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">DPBS without calcium and magnesium</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td></td>
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			<td height="21" style="height:21px;">Percoll</td>
			<td>GE Healthcare</td>
			<td>17-5445-02</td>
			<td>For density gradient centrifugation</td>
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		<tr height="21">
			<td height="21" style="height:21px;">GentleMACS &#8482; C tube&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-096-334</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GentleMACS dissociator device</td>
			<td>Miltenyi Biotec</td>
			<td>130-093-235</td>
			<td>Use the program SPLEEN_04</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Cell activation cocktail (with Brefeldin A)</td>
			<td>Biolegend</td>
			<td>423303</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-CD16/32</td>
			<td>eBioscience</td>
			<td>14-0161-81</td>
			<td>dilute 1:100</td>
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		<tr height="21">
			<td height="21" style="height:21px;">LIVE/DEAD fixable violet dead cell stain kit</td>
			<td>Life Technologies</td>
			<td>L34955</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Transcription factor buffer set</td>
			<td>BD Pharmingen</td>
			<td>562725</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">OneComp eBeads&#160;</td>
			<td>eBioscience</td>
			<td>01-1111-42</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">123 count eBeads</td>
			<td>eBioscience</td>
			<td>01-1234-42</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">CD45 AmCyan (clone 30-F11)</td>
			<td>BioLegend</td>
			<td>103138</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">CD3 PerCP-Cy5.5 (clone 17A2)</td>
			<td>BioLegend</td>
			<td>100218</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IL-17A FITC (clone TC11-18H10.1)</td>
			<td>BioLegend</td>
			<td>506910</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IL-17F APC (clone 9D3.1C8)</td>
			<td>BioLegend</td>
			<td>517004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD4 APC-Cy7 (clone GK1.5)</td>
			<td>BD Biosciences</td>
			<td>560181</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD8 APC (clone 53-67)</td>
			<td>eBioscience</td>
			<td>17-0081-82</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T-bet PE-Cy7 (clone 4B10)</td>
			<td>BioLegend</td>
			<td>644823</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IFN&#947; FITC (clone XMG1.2)</td>
			<td>BD Biosciences</td>
			<td>557724</td>
			<td></td>
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intracellular staining and flow cytometry to identify lymphocyte subsets within murine aorta, kidney and lymph nodes in a model of hypertension

It is now well known that T lymphocytes play a critical role in the development of several cardiovascular diseases1,2,3,4,5. For example, studies from our group have shown that hypertension is associated with an excessive accumulation of T cells in the vessels and kidney during the development of experimental hypertension6. Once in these tissues, T cells produce several cytokines that affect both vascular and renal function leading to vasoconstriction and sodium and water retention1,2. To fully understand how T cells cause cardiovascular and renal diseases, it is important to be able to identify and quantify the specific T cell subsets present in these tissues. T cell subsets are defined by a combination of surface markers, the cytokines they secrete, and the transcription factors they express. The complexity of the T cell population makes flow cytometry and intracellular staining an invaluable technique to dissect the phenotypes of the lymphocytes present in tissues. Here, we provide a detailed protocol to identify the surface and intracellular markers (cytokines and transcription factors) in T cells isolated from murine kidney, aorta and aortic draining lymph nodes in a model of angiotensin II induced hypertension. The following steps are described in detail: isolation of the tissues, generation of the single cell suspensions, ex vivo stimulation, fixation, permeabilization and staining. In addition, several fundamental principles of flow cytometric analyses including choosing the proper controls and appropriate gating strategies are discussed.

协议中所述的许可表面和细胞内标志物，从小鼠肾脏，主动脉及主动脉淋巴结在血管紧张素Ⅱ引起的高血压模型中分离的T细胞识别。代表性的结果介绍如下。 图1显示了用于从WT小鼠与血管紧张素II输注以诱导高血压的主动脉制得的单细胞悬液，以确定T细胞群体的门控策略。类似的策略在肾脏和淋巴结中。的前向散射区域（FSC-A）和侧散射区（SSC-A）的电压调节到检测白细胞种群（G1）和排除碎片。活细胞（G2）是阴性用于与太平洋蓝共轭的生存能力标记。那么活细胞门控前向散射高度（FSC-H）和前向散射面积（FSC-A）到门只在单人口（G3）。白细胞然后通过门控SSC-A和CD45（G4）中选择。 CD45是在本实施例中结合有AmCyan。最后，T细胞被门控为CD45 + CD3 +人口（G5）。 CD3是在本实施例中结合有PerCP-经Cy5.5。 测定IL-17A和IL-17F产生在该模型小鼠肾脏和主动脉内的T细胞的存在，单细胞悬浮液被染色对IL-17A和IL-17F。 图2示出荧光减去对IL-17A和一个肾脏样本中的IL-17F染色人控制（FMOs）（分别缀合用FITC和APC）。 FMO控件是包含所有在面板除了一个抗体的样品。如预期，很少细胞是阳性的FMO对照的IL-17A（ 图2A）或IL-17F（ 图2B）。这些控件允许阳性与阴性信号进行精确判别正确形容词乌斯栅极识别实验样品在正对IL-17A或IL-17F的细胞。 图3提供了从肾脏（ 图3A）分离的T细胞和用载体（假手术）或血管紧张素II（ANG II）的输注WT小鼠的主动脉（ 图3B）的细胞内染色的一个实例。的确，表达IL-17A或IL-17F的T细胞的子集可在该模型两种组织进行检测。 图4示出在这个模型中（ 图4A）主动脉引流淋巴结内的T细胞的细胞内染色。 CD8 + T细胞被第一选通（ 图4B）。然后，两个Tc1的标志物的表达，细胞因子IFNγ或转录因子T-bet的，从该CD8 + T细胞亚群定量。 图4C示出了用于干扰素的FMOs47;和淋巴结样品中的T-赌注染色（分别结合有FITC和PE-Cy7的）。 图4D显示了在WT小鼠的主动脉淋巴结输注血管紧张素II，CD8 +IFNγ+和CD8 + T赌注+细胞群可以被识别。 <img alt="图1" src="/files/ftp_upload/55266/55266fig1.jpg" /> 图1:流式细胞仪门的战略，以确定T细胞。白细胞第一门上的前向散射/侧向散射（FSC-A / SSC-A）散点图（G1），和活细胞被选中（G2）。然后将细胞选通单峰（FSC-A / FSC-H）（G3）。从G3细胞通过CD45（G4）中的表达进一步表征。最后，T细胞被门上的CD45 + CD3 +双阳性细胞（G5）。此单细胞悬浮液从一个野生型分离整个主动脉制备（WT）小鼠注入了angiot<html> ensin二诱发高血压。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55266/55266fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/55266/55266fig2.jpg" /> 图2:荧光减一对照（FMO）对IL-17A和IL-17F。 （A）中从血管紧张素II处理的鼠肾样品中分离的单个细胞悬浮液使用可固定生存能力标记（太平洋蓝），CD45（AmCyan），CD3（PerCP-经Cy5.5）和IL-17F（APC）染色。对IL-17A的抗体省略，以确定对IL-17A的适当的门控。 （B）的单细胞悬浮液从血管紧张素II处理的鼠肾样品中分离，使用一个可固定生存能力标记（太平洋蓝），CD45（AmCyan），CD3（PerCP-经Cy5.5）和IL-17A（FITC）染色。在这种情况下，对IL-17F的抗体被删去。urce.jove.com/files/ftp_upload/55266/55266fig2large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/55266/55266fig3.jpg" /> 图3:来自鼠肾和主动脉中分离的IL-17A和IL-17F的T细胞的细胞内染色。流式细胞点图表示来自用媒介物（假手术）输注WT小鼠或血管紧张素II（ANG II）和先前选通CD45 + CD3肾（A）和主动脉（B）在T细胞中的IL-17A和IL-17F表达+细胞。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55266/55266fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/55266/55266fig4.jpg" /> 图4:T细胞的细胞内染色<html> ■从鼠主动脉引流淋巴结中分离。 （ 一 ）宏观的WT鼠标2腰椎主动脉引流淋巴结的外观。 （B）的代表性的流式细胞术从一个野生型小鼠中分离4主动脉淋巴结分离的CD4 +和CD8 + T细胞的散点图输注血管紧张素II和先前选通的 CD45 + CD3 +细胞。 （C）从淋巴结中分离的单细胞悬浮液用活力标记（太平洋蓝）染色，CD3（PerCP-经Cy5.5），CD4（APC-Cy7的），和CD8（APC）。 FMO对照示于其中用于IFNγ（FITC）或T-bet的（PE-Cy7的）的抗体被省略，以确定正确的门控。 （ 四 ）流式细胞仪散点图证明使用由FMO控制确定的大门CD8 +淋巴结T细胞内IFNγ阳性和T-bet的表达。G"目标="_空白"&gt;点击此处查看该图的放大版本。

Watch this Scientific Journal Video about Intracellular Staining and Flow Cytometry to Identify Lymphocyte Subsets within Murine Aorta, Kidney and Lymph Nodes in a Model of Hypertension at JoVE.com

Intracellular Staining and Flow Cytometry to Identify Lymphocyte Subsets within Murine Aorta, Kidney and Lymph Nodes in a Model of Hypertension

本文提供了详细的方法来识别和量化细胞内染色鼠肾，主动脉和淋巴结中存在功能性T淋巴细胞亚群和流式细胞仪。被选中的血管紧张素II诱导高血压模型来解释，一步一步的程序和流式细胞仪的基本原则和细胞内染色。

细胞内染色和流式细胞仪识别鼠主动脉，肾脏和淋巴结内淋巴细胞亚群的高血压模型

It is now well known that T lymphocytes play a critical role in the development of several cardiovascular diseases1,2,3,4,5. For example, studies from our ...

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18.1K 次观看.  Vanderbilt University Medical Center. 本文提供了详细的方法来识别和量化细胞内染色鼠肾，主动脉和淋巴结中存在功能性T淋巴细胞亚群和流式细胞仪。被选中的血管紧张素II诱导高血压模型来解释，一步一步的程序和流式细胞仪的基本原则和细胞内染色。 

视频: Intracellular Staining and Flow Cytometry to Identify Lymphocyte Subsets within Murine Aorta, Kidney and Lymph Nodes in a Model of Hypertension

Non-surgical Intratracheal Instillation of Mice with Analysis of Lungs and Lung Draining Lymph Nodes by Flow Cytometry

Phagocytic cells such as alveolar macrophages and lung dendritic cells (LDCs) continuously sample antigens from the alveolar spaces in the 
lungs. LDCs, in particular, are known to migrate to the lung draining lymph nodes (LDLNs) where they present inhaled antigens to T cells initiating an 
appropriate immune response to a variety of immunogens1,2. To model interactions between the lungs and airborne antigens in mice, antigens can be 
administered intranasally1,3,4, intratracheally5 or as aerosols6. Delivery by each route involves distinct technical skills and limitations that need to be 
considered before designing an experiment. For example, intranasal and aerosolized exposure delivers antigens to both the lungs and the upper respiratory 
tract. Hence antigens can access the nasal associated lymphoid tissue (NALT)7, potentially complicating interpretation of the results. In addition, swallowing, 
sneezing and the breathing rate of the mouse may also lead to inconsistencies in the doses delivered. Although the involvement of the upper respiratory tract may 
be preferred for some studies, it can complicate experiments focusing on events specifically initiated in the lungs. In this setting, the intratracheal (i.t) 
route is preferable as it delivers test materials directly into the lungs and bypasses the NALT. Many i.t injection protocols involve either blind intubation of the 
trachea through the oral cavity or surgical exposure of the trachea to access the lungs. Herein, we describe a simple, consistent, non-surgical method for i.t 
instillation. The opening of the trachea is visualized using a laryngoscope and a bent gavage needle is then inserted directly into the trachea to deliver the 
innoculum. We also describe procedures for harvesting and processing of LDLNs and lungs for analysis of antigen trafficking by flow cytometry.

We illustrate non-surgical delivery of test materials into the lungs of anesthetized mice via the trachea. This method permits lung exposure to bacterial and viral pathogens, cytokines, antibodies, beads, chemicals, or dyes. We further describe harvesting and processing of lungs and lung draining lymph nodes (LDLNs) for flow cytometry.

非手术治疗的小鼠气管内滴入肺和肺淋巴结流式细胞仪分析

Phagocytic cells such as alveolar macrophages and lung dendritic cells (LDCs) continuously sample antigens from the alveolar spaces in the 
lungs. LDCs, ...

科研

免疫与感染

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 ...

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 ...

Isolation of Murine Lymph Node Stromal Cells

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host. 
Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.

Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells.

鼠淋巴结基质细胞的分离

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and ...

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

Granulocytes play a key role in the body&#8217;s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte function, in general these are limited in terms of the information they can provide. For example, most existing assays merely provide a percentage of how many granulocytes are activated following a single, fixed length incubation. Complicating matters, most assays focus on only one aspect of function due to limitations in detection technology. This report demonstrates a technique for simultaneous measurement of granulocyte phagocytosis of bacteria and oxidative burst. By measuring both of these functions at the same time, three unique phenotypes of activated granulocytes were identified: 1) Low Activation (minimal phagocytosis, no oxidative burst), 2) Moderate Activation (moderate phagocytosis, some oxidative burst, but no co-localization of the two functional events), and 3) High Activation (high phagocytosis, high oxidative burst, co-localization of phagocytosis and oxidative burst). A fourth population that consisted of inactivated granulocytes was also identified. Using assay incubations of 10, 20, and 40-min the effect of assay incubation duration on the redistribution of activated granulocyte phenotypes was assessed. A fourth incubation was completed on ice as a control. By using serial time incubations, the assay may be able to able to detect how a treatment spatially affects granulocyte function. All samples were measured using an image-based flow cytometer equipped with a quantitative imaging (QI) option, autosampler, and multiple lasers (488, 642, and 785 nm).

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

This method demonstrates a technique for assessing granulocyte function by simultaneously measuring phagocytosis of bacteria and oxidative burst. Image-based flow cytometry allowed for the identification of three distinct subsets of activated granulocytes that all differed in their relative functional capacity.

基于图像的流式细胞仪技术来评估变化粒细胞功能<em&gt;在体外</em

Granulocytes play a key role in the body&#8217;s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte ...

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal disease. Macrophages are heterogeneous cells that have been implicated in kidney injury. Macrophages may be classified into two different phenotypes: classically activated macrophages (M1 macrophages), that release pro-inflammatory cytokines and promote fibrosis; and alternatively activated macrophages (M2 macrophages) that are associated with immunoregulatory and tissue-remodeling functions. These macrophage phenotypes need to be discriminated and analyzed to determine their contribution to renal injury. However, there are scarce studies reporting consistent phenotypic and functional information about macrophage subtypes in inflammatory renal disease models, especially in rats. This fact may be related to the limited macrophage markers used in rats, contrary to mice. Therefore, novel strategies are necessary to quantify and characterize the renal content of these infiltrating cells in a reliable way. This manuscript details a protocol for kidney digestion and further phenotypic and quantitative analysis of macrophages from rat kidneys by flow cytometry. Briefly, kidneys were incubated with collagenase and total macrophages were identified according to the dual presence of CD45 (leukocytes common antigen) and CD68 (PAN macrophage marker) in live cells.This was followed by surface staining of CD86 (M1 marker) and CD163 (M2 marker). Rat peritoneal macrophages were used as positive control for macrophage marker detection by flow cytometry. Our protocol resulted in low cellular mortality and allowed characterization of different intracellular and surface protein markers, thus limiting the loss of cellular integrity observed in other protocols. Moreover, this procedure allows the use of macrophages for further techniques, including cell sorting and mRNA or protein expression studies, among others.

This manuscript describes a detailed protocol for phenotypic and quantitative analysis of resident macrophages from rat kidneys by flow cytometry. The resulting stained cells can be also used for other applications, including cell sorting, gene expression analysis or functional studies, thus increasing the information obtained in the experimental model.

通过流式细胞仪大鼠肾脏巨噬细胞的表型特征

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph node (DLN). The mobilization of DCs to the DLN is complex and remains to be fully elucidated during infection. Herein described is the use of an innovative, simple assay that relies on the fluorochrome 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) to track the migration of DCs during footpad infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin (BCG) in C57BL/6 mice. This assay enables the characterization of skin DC sub-populations that actively relocate to the draining, popliteal LN in response to BCG. This protocol originates from a BCG model where migratory skin DCs were identified by flow cytometry. The assay is amiable to the study and identification of DCs or other cells that home to the popliteal LN after inoculation of microbes, their metabolites or other inflammatory stimuli in the footpad, and consequently to study factors that regulate the migration of these cells.

An assay in mice to track cell migration from skin to draining lymph node is described which enables the characterization of skin Dendritic cells mobilized to the lymph node after footpad infection with Bacille Calmette-Gu&#233;rin.

基于CFSE-试验鼠皮肤树突状细胞的迁移习进引流淋巴结在与感染<em&gt;牛分枝杆菌</em&gt;卡介苗

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph ...

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) cycles between a tsetse vector and a mammalian host. It evades the mammalian host immune system by periodically switching the dense, variant surface glycoprotein (VSG) that covers its surface. The detection of antigenic variation in Trypanosoma brucei can be both cumbersome and labor intensive. Here, we present a method for quantifying the number of parasites that have 'switched' to express a new VSG in a given population. The parasites are first stained with an antibody against the starting VSG, and then stained with a secondary antibody attached to a magnetic bead. Parasites expressing the starting VSG are then separated from the rest of the population by running the parasites over a column attached to a magnet. Parasites expressing the dominant, starting VSG are retained on the column, while the flow-through contains parasites that express a new VSG as well as some contaminants expressing the starting VSG. This flow-through population is stained again with a fluorescently labeled antibody against the starting VSG to label contaminants, and propidium iodide (PI), which labels dead cells. A known number of absolute counting beads that are visible by flow cytometry are added to the flow-through population. The ratio of beads to number of cells collected can then be used to extrapolate the number of cells in the entire sample. Flow cytometry is used to quantify the population of switchers by counting the number of PI negative cells that do not stain positively for the starting, dominant VSG. The proportion of switchers in the population can then be calculated using the flow cytometry data.

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

African trypanosomes grown in vitro undergo antigenic variation at a low rate, such that populations are made up of parasites expressing a dominant variant surface glycoprotein (VSG) type and a small population of "switched" variants. This protocol describes a fast method for detecting and quantifying these populations.

检测<em&gt;布氏锥虫</em&gt;变体表面糖蛋白通过磁性细胞分选和流式细胞仪开关

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) ...

Utilization of Capsules for Negative Staining of Viral Samples within Biocontainment

Transmission electron microscopy (TEM) is used to observe the ultrastructure of viruses and other microbial pathogens with nanometer resolution. Most biological materials do not contain dense elements capable of scattering electrons to create an image; therefore, a negative stain, which places dense heavy metal salts around the sample, is required. In order to visualize viruses in suspension under the TEM they must be applied to small grids coated with a transparent surface only nanometers thick. Due to their small size and fragility, these grids are difficult to handle and easily moved by air currents. The thin surface is easily damaged, leaving the sample difficult or impossible to image. Infectious viruses must be handled in a biosafety cabinet (BSC) and some require a biocontainment laboratory environment. Staining viruses in biosafety levels (BSL)-3 and -4 is especially challenging because these environments are more turbulent and technicians are required to wear personal protective equipment (PPE), which decreases dexterity. 
In this study, we evaluated a new device to assist in negative staining viruses in biocontainment. The device is a capsule that works as a specialized pipette tip. Once grids are loaded into the capsule, the user simply aspirates reagents into the capsule to deliver the virus and stains to the encapsulated grid, thus eliminating user handling of grids. Although this technique was designed specifically for use in BSL-3 or -4 biocontainment, it can ease sample preparation in any lab environment by enabling easy negative staining of virus. This same method can also be applied to prepare negative stained TEM specimens of nanoparticles, macromolecules and similar specimens.

This protocol provides instruction for negative staining virus samples which can easily be used in BSL-2, -3, or -4 laboratories. It includes the use of an innovative processing capsule, which protects the transmission electron microscopy grid and provides the user easier handling in the more turbulent environments within biocontainment.

利用胶囊对生物达标中病毒样品的阴性染色

Transmission electron microscopy (TEM) is used to observe the ultrastructure of viruses and other microbial pathogens with nanometer resolution. Most ...

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 ...