Name: intracellular staining and flow cytometry to identify lymphocyte subsets within murine aorta, kidney and lymph nodes in a model of hypertension
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Description: 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

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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R. <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.">Single-cell genomics.</a> Nat Methods. 8 (4), 311-314 (2011).</li><li>Chattopadhyay, P. 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。

<table><tbody><tr><td>Collagenase D</td><td>ROCHE</td><td>11088882001</td><td></td></tr><tr><td>Collagenase A</td><td>ROCHE</td><td>10103586001</td><td></td></tr><tr><td>Collagenase B</td><td>ROCHE</td><td>11088815001</td><td></td></tr><tr><td>Dnase</td><td>ROCHE</td><td>10104159001</td><td></td></tr><tr><td>1x Red blood cell lysis buffer</td><td>eBioscience</td><td>00-4333-57</td><td></td></tr><tr><td>RPMI Medium 1614 1x</td><td>Gibco</td><td>11835-030</td><td></td></tr><tr><td>DPBS without calcium and magnesium</td><td>Gibco</td><td>14190-144</td><td></td></tr><tr><td>Percoll</td><td>GE Healthcare</td><td>17-5445-02</td><td>For density gradient centrifugation</td></tr><tr><td>GentleMACS&amp;trade; C tube</td><td>Miltenyi Biotec</td><td>130-096-334</td><td></td></tr><tr><td>GentleMACS dissociator device</td><td>Miltenyi Biotec</td><td>130-093-235</td><td>Use the program SPLEEN_04</td></tr><tr><td>Cell activation cocktail (with Brefeldin A)</td><td>Biolegend</td><td>423303</td><td></td></tr><tr><td>anti-CD16/32</td><td>eBioscience</td><td>14-0161-81</td><td>dilute 1:100</td></tr><tr><td>LIVE/DEAD fixable violet dead cell stain kit</td><td>Life Technologies</td><td>L34955</td><td></td></tr><tr><td>Transcription factor buffer set</td><td>BD Pharmingen</td><td>562725</td><td></td></tr><tr><td>OneComp eBeads&amp;nbsp;</td><td>eBioscience</td><td>01-1111-42</td><td></td></tr><tr><td>123 count eBeads</td><td>eBioscience</td><td>01-1234-42</td><td></td></tr><tr><td>CD45 AmCyan (clone 30-F11)</td><td>BioLegend</td><td>103138</td><td></td></tr><tr><td>CD3 PerCP-Cy5.5 (clone 17A2)</td><td>BioLegend</td><td>100218</td><td></td></tr><tr><td>IL-17A FITC (clone TC11-18H10.1)</td><td>BioLegend</td><td>506910</td><td></td></tr><tr><td>IL-17F APC (clone 9D3.1C8)</td><td>BioLegend</td><td>517004</td><td></td></tr><tr><td>CD4 APC-Cy7 (clone GK1.5)</td><td>BD Biosciences</td><td>560181</td><td></td></tr><tr><td>CD8 APC (clone 53-67)</td><td>eBioscience</td><td>17-0081-82</td><td></td></tr><tr><td>T-bet PE-Cy7 (clone 4B10)</td><td>BioLegend</td><td>644823</td><td></td></tr><tr><td>IFN&amp;gamma; FITC (clone XMG1.2)</td><td>BD Biosciences</td><td>557724</td><td></td></tr></tbody></table>

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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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 ...

intracellular-staining-flow-cytometry-to-identify-lymphocyte-subsets

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

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

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

Quantitative Analysis and Characterization of Atherosclerotic Lesions in the Murine Aortic Sinus

Atherosclerosis is a disease of the large arteries and a major underlying cause of myocardial infarction and stroke. Several different mouse models have been developed to facilitate the study of the molecular and cellular pathophysiology of this disease. In this manuscript we describe specific techniques for the quantification and characterization of atherosclerotic lesions in the murine aortic sinus and ascending aorta. The advantage of this procedure is that it provides an accurate measurement of the cross-sectional area and total volume of the lesion, which can be used to compare atherosclerotic progression across different treatment groups. This is possible through the use of the valve leaflets as an anatomical landmark, together with careful adjustment of the sectioning angle. We also describe basic staining methods that can be used to begin to characterize atherosclerotic progression. These can be further modified to investigate antigens of specific interest to the researcher. The described techniques are generally applicable to a wide variety of existing and newly created dietary and genetically-induced models of atherogenesis.

We describe procedures to quantify and characterize atherosclerotic lesions in mouse models using precision sectioning of the aortic sinus and ascending aorta combined with histochemical and immunohistochemical analysis.

Atherosclerosis is a disease of the large arteries and a major underlying cause of myocardial infarction and stroke. Several different mouse models have ...

医学

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 ...

Visualizing Leukocyte Rolling and Adhesion in Angiotensin II-Infused Mice: Techniques and Pitfalls

Epifluorescence intravital video microscopy (IVM) of blood vessels is an established method to evaluate the activation of immune cells and their ability to role and adhere to the endothelial layer. Visualization of circulating cells by injection of fluorescent dyes or fluorophore-coupled antibodies is commonly used. Alternatively, fluorescent reporter mice can be used. Interactions of leukocytes, in particular lysozyme M+ (LysM+) monocytes, with the vessel wall play pivotal roles in promoting vascular dysfunction and arterial hypertension. We here present the technique to visualize and quantify leukocyte rolling and adhesion in carotid arteries in angiotensin II (AngII)-induced hypertension in mice by IVM.
The implantation of a catheter damages the vascular wall and leads to altered blood cell responses. We compared different injection techniques and administration routes to visualize leukocytes in a LysMCre+IRG+ mouse with widespread expression of red fluorescent protein and conditional expression of green fluorescent protein in LysM+ cells. To study LysM+ cell activation, we used AngII infused mice in which rolling and adhesion of leukocytes to the endothelium is increased. We either injected acridine orange using a jugular catheter or directly though the tail vein and compared the amount of rolling and adhering cells. We found that jugular catheter implantation per se increased the number of rolling and adhering LysM+ cells in sham-infused LysMCre+IRG+ mice compared to controls. This activation was augmented in AngII-infused mice. Interestingly, injecting acridine orange directly through the tail vein did not increase LysM+ cell adhesion or rolling in sham-infused mice. We thereby demonstrated the importance of transgenic reporter mice expressing fluorescent proteins to not interfere with in vivo processes during experimentation. Furthermore, tail vein injection of fluorescent tracers might be a possible alternative to jugular catheter injections.

This manuscript describes the use of transgenic reporter mice and different administration routes of fluorescent dyes in angiotensin II-induced hypertension using intravital video microscopy of blood vessels to evaluate the activation of immune cells and their ability to roll and adhere to the endothelium.

血管紧张素 II. 型小鼠白细胞滚动和粘附的可视化技术及缺陷

Epifluorescence intravital video microscopy (IVM) of blood vessels is an established method to evaluate the activation of immune cells and their ability ...

Digestion of the Murine Liver for a Flow Cytometric Analysis of Lymphatic Endothelial Cells

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the lymph nodes where cellular debris and antigens can be surveyed. We are very interested in understanding how the lymphatic vasculature might be involved in inflammation and immune cell function within the liver. However, very little has been published establishing digestion protocols for the isolation of lymphatic endothelial cells (LECs) from the liver or specific markers that can be used to evaluate liver LECs on a per cell basis. Therefore, we optimized a method for the digestion and staining of the liver in order to evaluate the LEC population in the liver. We are confident that the method outlined here will be useful for the identification and isolation of LECs from the liver and will strengthen our understanding of how LECs respond to the liver microenvironment.

The goal of this protocol is to identify lymphatic endothelial cell populations within the liver using described markers. We utilize collagenase IV and DNase and a gentle mincing of tissue, combined with flow cytometry, to identify a distinct population of lymphatic endothelial cells.

小鼠肝脏消化对淋巴内皮细胞的流式细胞仪分析

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the ...

Quantification of Atherosclerosis in Mice

Cardiovascular disease is the main cause of death in the world. The underlying cause in most cases is atherosclerosis, which is in part a chronic inflammatory disease. Experimental atherosclerosis studies have elucidated the role of cholesterol and inflammation in the disease process. This has led to successful clinical trials with pharmaceutical agents that reduce clinical manifestations of atherosclerosis. Careful and well-controlled experiments in mouse models of the disease could further elucidate the pathogenesis of the disease, which is not fully understood. Standardized lesion analysis is important to reduce experimental variability and increase reproducibility. Determining lesion size in aortic root, aortic arch, and brachiocephalic artery are common endpoints in experimental atherosclerosis. This protocol provides a technical description for evaluation of atherosclerosis at all these sites in a single mouse. The protocol is particularly useful when material is limited, as is frequently the case when genetically modified animals are being characterized.

Murine models of atherosclerosis are useful tools to investigate pathogenic pathways on a molecular level, but require standardized quantification of lesion development. This protocol describes an optimized method to determine lesion size in the major arterial vessels including the aortic root, aortic arch, and brachiocephalic artery.

小鼠动脉粥样硬化的定量

Cardiovascular disease is the main cause of death in the world. The underlying cause in most cases is atherosclerosis, which is in part a chronic ...

Isolation of Mouse Kidney-Resident CD8+ T cells for Flow Cytometry Analysis

Tissue-resident memory T cell (TRM) is a rapidly expanding field of immunology research. Isolating T cells from various non-lymphoid tissues is one of the key steps to investigate TRMs. There are slight variations in lymphocyte isolation protocols for different organs. Kidney is an essential non-lymphoid organ with numerous immune cell infiltration especially after pathogen exposure or autoimmune activation. In recent years, multiple labs including our own have started characterizing kidney resident CD8+ T cells in various physiological and pathological settings in both mouse and human. Due to the abundance of T lymphocytes, kidney represents an attractive model organ to study TRMs in non-mucosal or non-barrier tissues. Here, we will describe a protocol commonly used in TRM-focused labs to isolate CD8+ T cells from mouse kidneys following systemic viral infection. Briefly, using an acute lymphocytic choriomeningitis virus (LCMV) infection model in C57BL/6 mice, we demonstrate intravascular CD8+ T cell labeling, enzymatic digestion, and density gradient centrifugation to isolate and enrich lymphocytes from mouse kidneys to make samples ready for the subsequent flow cytometry analysis.

Isolation of Mouse Kidney-Resident CD8+ T cells for Flow Cytometry Analysis

Following viral infection, kidney harbors a relatively large number of CD8+ T cells and offers an opportunity to study non-mucosal TRM cells. Here, we describe a protocol to isolate mouse kidney lymphocytes for flow cytometry analysis.

小鼠肾-居民CD8+T 细胞的分离，用于流细胞测量分析

Tissue-resident memory T cell (TRM) is a rapidly expanding field of immunology research. Isolating T cells from various non-lymphoid tissues is one of the ...

Quantitative Analysis of Cellular Composition in Advanced Atherosclerotic Lesions of Smooth Muscle Cell Lineage-Tracing Mice

Atherosclerosis remains the leading cause of death worldwide and, despite countless preclinical studies describing promising therapeutic targets, novel interventions have remained elusive. This is likely due, in part, to a reliance on preclinical prevention models investigating the effects of genetic manipulations or pharmacological treatments on atherosclerosis development rather than the established disease. Also, results of these studies are often confounding because of the use of superficial lesion analyses and a lack of characterization of lesion cell populations. To help overcome these translational hurdles, we propose an increased reliance on intervention models that employ investigation of changes in cellular composition at a single cell level by immunofluorescent staining and confocal microscopy. To this end, we describe a protocol for testing a putative therapeutic agent in a murine intervention model including a systematic approach for animal dissection, embedding, sectioning, staining, and quantification of brachiocephalic artery lesions. In addition, due to the phenotypic diversity of cells within late-stage atherosclerotic lesions, we describe the importance of using cell-specific, inducible lineage tracing mouse systems and how this can be leveraged for unbiased characterization of atherosclerotic lesion cell populations. Together, these strategies may assist vascular biologists to more accurately model therapeutic interventions and analyze atherosclerotic disease and will hopefully translate into a higher rate of success in clinical trials.

We propose a standardized protocol to characterize the cellular composition of late-stage murine atherosclerotic lesions including systematic methods of animal dissection, tissue embedding, sectioning, staining, and analysis of brachiocephalic arteries from atheroprone smooth muscle cell lineage tracing mice.

平滑肌细胞近系追踪小鼠晚期动脉粥样硬化病变细胞成分的定量分析

Atherosclerosis remains the leading cause of death worldwide and, despite countless preclinical studies describing promising therapeutic targets, novel ...

Flow Cytometry Analysis of Immune Cell Subsets within the Murine Spleen, Bone Marrow, Lymph Nodes and Synovial Tissue in an Osteoarthritis Model

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent evidence indicates a potential inflammatory component of the disease, with both T-cells and monocytes/macrophages potentially associated with the pathogenesis of OA. Further studies postulated an important role for subsets of both inflammatory cell lineages, such as Th1, Th2, Th17, and T-regulatory lymphocytes, and M1, M2, and synovium-tissue-resident macrophages. However, the interaction between the local synovial and systemic inflammatory cellular response and the structural changes in the joint is unknown. To fully understand how T-cells and monocytes/macrophages contribute towards OA, it is important to be able to quantitively identify these cells and their subsets simultaneously in synovial tissue, secondary lymphatic organs and systemically (the spleen and bone marrow). Nowadays, the different inflammatory cell subsets can be identified by a combination of cell-surface markers making multi-color flow cytometry a powerful technique in investigating these cellular processes. In this protocol, we describe detailed steps regarding the harvest of synovial tissue and secondary lymphatic organs as well as generation of single cell suspensions. Furthermore, we present both an extracellular staining assay to identify monocytes/macrophages and their subsets as well as an extra- and intra-cellular staining assay to identify T-cells and their subsets within the murine spleen, bone marrow, lymph nodes and synovial tissue. Each step of this protocol was optimized and tested, resulting in a highly reproducible assay that can be utilized for other surgical and non-surgical OA mouse models.

Here, we describe a detailed and reproducible flow cytometry protocol to identify monocyte/macrophage and T-cell subsets using both extra- and intracellular staining assays within the murine spleen, bone marrow, lymph nodes and synovial tissue, utilizing an established surgical model of murine osteoarthritis.

骨关节炎模型中骨髓、骨髓、淋巴结和合成组织内免疫细胞子集的流动细胞测量分析

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent ...

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on changes in intracellular calcium ([Ca2+]i) and nitric oxide (NO). In order to understand how [Ca2+]i, NO and downstream molecules are handled by a blood vessel in response to vasoconstrictors and vasodilators, we developed a novel technique that applies calcium-labeling (or NO-labeling) dyes with two photon microscopy to measure calcium handling (or NO production) in isolated blood vessels. Described here is a detailed step-by-step procedure that demonstrates how to isolate an aorta from a rat, label calcium or NO within the endothelial or smooth muscle cells, and image calcium transients (or NO production) using a two photon microscope following physiological or pharmacological stimuli. The benefits of using the method are multi-fold: 1) it is possible to simultaneously measure calcium transients in both endothelial cells and smooth muscle cells in response to different stimuli; 2) it allows one to image endothelial cells and smooth muscle cells in their native setting; 3) this method is very sensitive to intracellular calcium or NO changes and generates high resolution images for precise measurements; and 4) described approach can be applied to the measurement of other molecules, such as reactive oxygen species. In summary, application of two photon laser emission microscopy to monitor calcium transients and NO production in the endothelial and smooth muscle cells of an isolated blood vessel has provided high quality quantitative data and promoted our understanding of the mechanisms regulating vascular function.

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Vascular cell functiondepends on activity of intracellular messengers. Described here is an ex vivo two photon imaging method that allows the measurement of intracellular calcium and nitric oxide levels in response to physiological and pharmacological stimuli in individual endothelial and smooth muscle cells of an isolated aorta.

细胞内钙的双光子成像<sup&gt; 2+</sup&gt;的离体大鼠主动脉的处理和一氧化氮的内皮细胞和平滑肌细胞

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on ...

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

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