This work is supported by a Research Grant from Dialysis Clinics Inc. and from the University of Missouri Research Board Grant.

执行的所有协议的步骤进行了审查和密苏里州的动物护理和使用委员会大学（ACUC）的批准。对于这个协议，年龄15周雄性C57BL / 6小鼠利用虽然理论上在任何年龄任何啮齿动物可以用于实验。因为，这是一个非存活手术，安乐死放血和双边气胸实现。 1.肾脏灌注注:器官如心脏，肝脏和肾脏灌注消除可能与数据的解释干扰血液。因此，如果可能，我们始终灌注器官。 <ol><li>鼠标放置在异氟醚（3毫升/分钟）感应腔室，直到它是不动的。接下来，取出鼠标移出腔，背其放在解剖台上，并将鼠标鼻异氟醚锥和1.5毫升/分钟通过调节器的速度推异氟醚。 </li><li>检查脚趾捏responsË用镊子来评估，如果动物已经取得了手术的麻醉平面。清洗用生理盐水腹侧腹部和切开用剪刀腹部和推腹部器官（出手术区域），以鼠标的左侧。 </li><li>收集从下腔静脉的血液用25号针头固定到一个1毫升注射器，并将其添加到含有10微升0.5M EDTA的1.5ml微量离心管中。 注:采血是可选的。等离子体可以用于生化测定。白细胞可以用于流式细胞仪分析。 </li><li>使用剪刀，使在右心房的小切口，以从下一步骤放出的血液和PBS的混合物。 </li><li>取50毫升注射器，用冰冷的PBS（20-30毫升）填充。接下来，附加一个21-25摹针注射器，穿刺在其顶点左心室（同时轻轻持物钳之间的心脏），并慢慢灌注超过课程磷酸盐缓冲液pH值7.4（PBS）左心室1-2分钟。 注意:心脏立即Blanches的经灌注的前几毫升。观察肝脏进行热烫，因为这表明灌注经由穿过腔静脉静脉回流引流血出肝脏。此外，确保，因为这表示右心室穿刺肺不输液期间扩大。如果肺是扩大，撤回针头稍稍向后进入左心室并继续灌注。从第一切口直到安乐死实现所花费的时间小于2分钟</li></ol> 2.肾解剖<ol><li>灌注完成后，用剪刀和镊子来剖析其附件（血管，脂肪和肾上腺）和消费右肾。 </li><li>轻轻地取出覆盖切割，并用钳子解剖肾囊中。称取肾。 注:称重肾脏是可选的。然而，一些实验可导致在肾尺寸/重量可被用于标准化每克组织的细胞产量的变化。 </li><li>称重肾脏后，将其放置在1ml​​含0.5％FBS的RPMI-1640或流式细胞术染色缓冲液（50ml锥形螺帽管），并放置该管在冰上直到进一步使用。 注意:为了便于描述，我们将描述在流式细胞染色缓冲液的流动的免疫细胞的分离的其余部分。此外，我们将描述的免疫细胞的分离从单一肾，虽然两个肾脏或肾脏的1 + 1/3 次可以根据下游实验（可选）被使用。 1/3 次肾脏的可冻结蛋白质/ RNA的实验和1/3 次可以放置在用于免疫组织化学或透射电子显微镜的缓冲区。 </li></ol> 3.肾同质化<ol><li>标签的5-80毫升容量的同质化袋样品识别信息，取出里面的过滤器（STR）并丢弃。用5ml冰冷的流式细胞术染色缓冲溶液填充袋转移肾到缓冲器。重复此过程的第二个样品或多个样品需要处理。 </li><li>对折的均质袋，使得一个半部具有在流式细胞术染色缓冲液浸没肾脏，而另一半被简单地折叠在它。 </li><li>打开组织匀浆并将其设置为快速（设置）的转速。将折叠的同质化袋肾脏面临的桨，接近底部边缘，但要确保袋子的下端不会滑动拨片的下边框的下方。 <ol><li>同时处理第二个样品，因为有2拨片。选择时间为120秒。观察桨来回移动均匀化肾脏。 注意:同时，该停机时间可用于制备2更肾（或其他组织）的样品，而前两个处理。 </li></OL&gt; </li><li>与第一组同质化完成后，将袋转移至冰上，并用下一个样本集等重装组织匀浆。目测确保肾脏充分混匀后，没有大块是可见的。 注意:通常，在快转的组织匀浆2分钟是足够的肾脏。 </li><li>放置一个100微米的细胞滤网入冰上50ml锥形管中。接着，完全上吸取匀浆至细胞过滤。在4℃下50×g离心1分钟设定的楼层离心机和样品装入离心机。 注意:离心将确保所有的匀浆（包含细胞）已经穿过细胞过滤通过。 </li><li>除去从离心机管，并将它们回冰上。丢弃电池滤网，保持匀浆。添加等体积（〜6ml）中1×RBC裂解缓冲液将细胞沉淀/上清液和移液器上下数次以使均匀的细胞悬浮液。 VisuaLLY，观察一些红色的立即中止减少。离开冰上管5分钟以​​达到最大的RBC裂解。 注意:如果研究者相信肾脏灌注和较少细胞操作期望的RBC裂解步骤可以跳过。我们已经通过不使细胞悬液RBC裂解观察肾细胞产率显著增加。 </li><li>旋管在已设定为500 XG，4℃5分钟，以形成细胞沉淀离心机。 注:在步骤3.6和3.7，胶体二氧化硅（珀）的密度梯度离心准备。 <ol><li>取10毫升圆底聚丙烯管，并与相应的样品号标记它们并将它们放置在一个管架。 注:以下介绍如何使足够的密度梯度试剂4肾脏样本。 </li><li>坐7毫升流式细胞术染色缓冲液并吸取入15ml锥形管中。加入1毫升的2M蔗糖准备0.25M蔗糖。 </li><li>接下来，我们7.2毫升密度梯度试剂吸管它到另外15毫升的锥形管。加1.55毫升的流式细胞术染色缓冲溶液和1.25毫升的2M蔗糖以制备72％的密度梯度试剂和0.25M蔗糖。 </li><li>吸管1毫升72％的密度梯度试剂成每个样本一个回合底聚丙烯管中。 </li></ol></li></ol> 4.梯度离心<ol><li>步骤3.7后，将管，轻轻倒出上清液以平滑的动作，也倒出而管倒持形成液体的下一个下降。 注意:此步骤是因为留下过量液体可以改变密度梯度足以创建在产量变化重要。当管被设置回直立，〜200-300微升的液体保持与细胞沉淀。 </li><li>吸管加入1ml 0.25M蔗糖到细胞沉淀并轻轻但大力吸管上下布林克细胞均匀的悬浮液。接着，将细胞悬浮液添加到1ml 72％的密度梯度试剂（3.7.4），并再次吸取剧烈混合细胞以形成36％的密度梯度试剂。 </li><li>取1毫升的72％的密度梯度试剂混入聚丙烯巴斯德（传送）吸移管。与灯泡连压（保持液柱的尖端，并防止气泡形成），插入巴斯德吸管向细胞悬浮液的底部（36％的密度梯度试剂）轻轻和卸载72％的密度梯度剂，以形成一个不同的重层。 </li><li>轻轻拿起样品管并放入离心机，其是在室温下此步骤。旋在500×g下20-30分钟。 </li><li>同时，准备新一轮的两套聚丙烯轮相应的样本标识底部的离心管。 </li><li>步骤4.4完成后，取出试样轻轻含聚丙烯管，以最小的抖动和设置下来在管架上。 </li><li>拿起第一样品管及其相应的空管。用聚丙烯巴斯德吸管，轻轻但彻底吸测量，去掉上面的"垃圾层"或"糊糊的材料"，并把球送入空圆底离心管中。 <ol><li>继续除去梯度的上部，直到"血沉棕黄层"达到（可能出现灰白色与发红[红细胞]）。停止抽吸如果从"血沉棕黄层"达到了〜3mm的高度。 注意:确保这东西是从梯度上部继续吸之前完全消失了。 </li></ol></li><li>取新鲜巴斯德吸管和一个新的圆底离心管中，并轻轻地取下捉鬼外套。继续吸3 -血沉棕黄层低于5毫米，以确保大部分的细胞被收集并加入到适当的取样标识符第2 次新圆底管中。 </li><li>加1-2毫升邻˚F流式细胞术染色缓冲液溶液，以第2 次管和通过上下吹打调匀。 </li><li>降速细胞在500×g离心5分钟，4℃。 A 2 次清洗步骤用流式细胞仪染色缓冲液是可选的。悬浮在500微升流式细胞仪染色缓冲溶液，并继续使用血球计数细胞。 </li></ol> 5.可选（细胞标记） <ol><li> Aliqout每个样本为相同数目的细胞。添加阻断抗体以每10 6个细胞0.5微克（抗CD16 / CD32的FcR）的稀释液，并在冰上孵育15分钟。 </li><li>添加被确定为彼此由研究者很好地工作，实际的实验之前，初级抗体（缀合到合适的荧光团）的期望的鸡尾酒。在冰上孵育30分钟。清洗细胞，用PBS。 注:对于肾虚的免疫细胞标记示范的这个手稿，我们使用了T-LYmphocyte和巨噬细胞/树突状细胞面板。 T淋巴细胞小组由可固定生存能力染色FVS510，抗CD45（克隆30-F11）的BV421，抗Foxp3的（克隆:FJK-16）APC，抗CD127（克隆:A7R34）PE / Cy7的，抗CD44（克隆:IM7）PerCP /经Cy5.5，抗CD4（克隆:RM4-5）APC-Cy7的，抗CD8（克隆:53-6.7）BV785。巨噬细胞小组由可固定生存能力染色FVS510，抗CD45（克隆30-F11）的BV421，抗Ly6G（克隆:1A8）FITC，抗CD11b（克隆:M1 / 70）PerCP-经Cy5.5，反F4 / 80（克隆:BM8）APC，抗CD11c（克隆:N418）BV785。 </li><li>添加可固定活力染色和台式离开5分钟。与洗净流式细胞仪染色缓冲液中的细胞，去除多余的可固定可行性污点。 </li><li>添加固定/通透试剂，并在4ºC孵育过夜。洗通透的解决方案。 </li><li>对于细胞内染色，用抗CD16 / 32的FcR再次阻塞10分钟。添加细胞内染色和孵育20-30分钟。洗2X在PErmeabilization解决方案。 </li><li>悬浮流式细胞仪染色缓冲液和运行样品通过流式细胞仪机3,9。 </li></ol>

<ol>
	<li>Ascon, D. B., 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=Phenotypic+and+functional+characterization+of+kidney-infiltrating+lymphocytes+in+renal+ischemia+reperfusion+injury.">Phenotypic and functional characterization of kidney-infiltrating lymphocytes in renal ischemia reperfusion injury.</a> J. Immunol. 177 (5), 3380-3387 (2006).</li><li>Ascon, M., 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=Renal+ischemia-reperfusion+leads+to+long+term+infiltration+of+activated+and+effector-memory+T+lymphocytes.">Renal ischemia-reperfusion leads to long term infiltration of activated and effector-memory T lymphocytes.</a> Kidney Int. 75 (5), 526-535 (2009).</li><li>Belliere, J., 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=Specific+macrophage+subtypes+influence+the+progression+of+rhabdomyolysis-induced+kidney+injury.">Specific macrophage subtypes influence the progression of rhabdomyolysis-induced kidney injury.</a> J. Am. Soc. Nephrol. 26 (6), 1363-1377 (2015).</li><li>Chen, Z., 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=Collagenase+digestion+down-regulates+the+density+of+CD27+on+lymphocytes.">Collagenase digestion down-regulates the density of CD27 on lymphocytes.</a> J. Immunol. Methods. 413, 57-61 (2014).</li><li>Dong, X., 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=Resident+dendritic+cells+are+the+predominant+TNF-secreting+cell+in+early+renal+ischemia-reperfusion+injury.">Resident dendritic cells are the predominant TNF-secreting cell in early renal ischemia-reperfusion injury.</a> Kidney Int. 71 (7), 619-628 (2007).</li><li>Hickey, F. B., Martin, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diabetic+kidney+disease+and+immune+modulation.">Diabetic kidney disease and immune modulation.</a> Curr. Opin. Pharmacol. , (2013).</li><li>Kawakami, T., 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=Resident+renal+mononuclear+phagocytes+comprise+five+discrete+populations+with+distinct+phenotypes+and+functions.">Resident renal mononuclear phagocytes comprise five discrete populations with distinct phenotypes and functions.</a> J. Immunol. 191 (6), 3358-3372 (2013).</li><li>Liu, X., 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=Isolating+glomeruli+from+mice:+A+practical+approach+for+beginners.">Isolating glomeruli from mice: A practical approach for beginners.</a> Exp. Ther. Med. 5 (5), 1322-1326 (2013).</li><li>Picot, J., Guerin, C. L., Le Van, K. C., Boulanger, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry:+retrospective,+fundamentals+and+recent+instrumentation.">Flow cytometry: retrospective, fundamentals and recent instrumentation.</a> Cytotechnology. 64 (2), 109-130 (2012).</li><li>Ricardo, S. D., van, G. H., Eddy, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+diversity+in+renal+injury+and+repair.">Macrophage diversity in renal injury and repair.</a> J. Clin. Invest. 118 (11), 3522-3530 (2008).</li><li>Rodriguez-Iturbe, B., Pons, H., Herrera-Acosta, J., Johnson, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+immunocompetent+cells+in+nonimmune+renal+diseases.">Role of immunocompetent cells in nonimmune renal diseases.</a> Kidney Int. 59 (5), 1626-1640 (2001).</li><li>Vielhauer, V., 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=Efficient+renal+recruitment+of+macrophages+and+T+cells+in+mice+lacking+the+duffy+antigen/receptor+for+chemokines.">Efficient renal recruitment of macrophages and T cells in mice lacking the duffy antigen/receptor for chemokines.</a> Am. J. Pathol. 175 (1), 119-131 (2009).</li><li>Weisheit, C. K., Engel, D. R., Kurts, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+Cells+and+Macrophages:+Sentinels+in+the+Kidney.">Dendritic Cells and Macrophages: Sentinels in the Kidney.</a> Clin. J. Am. Soc. Nephrol. , (2015).</li><li>Williams, S. K., McKenney, S., Jarrell, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagenase+lot+selection+and+purification+for+adipose+tissue+digestion.">Collagenase lot selection and purification for adipose tissue digestion.</a> Cell Transplant. 4 (3), 281-289 (1995).</li><li>Yamamoto, T., 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=Deterioration+and+variability+of+highly+purified+collagenase+blends+used+in+clinical+islet+isolation.">Deterioration and variability of highly purified collagenase blends used in clinical islet isolation.</a> Transplantation. 84 (8), 997-1002 (2007).</li><li>Zhou, J., Nagarkatti, P., Zhong, Y., Nagarkatti, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+modulation+by+chondroitin+sulfate+and+its+degraded+disaccharide+product+in+the+development+of+an+experimental+model+of+multiple+sclerosis.">Immune modulation by chondroitin sulfate and its degraded disaccharide product in the development of an experimental model of multiple sclerosis.</a> J. Neuroimmunol. 223 (1-2), 55-64 (2010).</li></ol>

作者什么都没有透露。

我们在这里提出了一个方法，以获得从肾脏的免疫细胞中的可靠和有效的方式。主要的修改的广泛使用的胶原酶消化步骤（组织机械破碎）保存约30分钟，并有大量活的免疫细胞的隔离下两个小时4肾脏样品花费。此外，根据我们的研究问题，我们现在只用一个单一的肾（其他肾脏可以用于通过Western印迹，免疫组织化学和mRNA分析通过PCR蛋白质分析）对我们的免疫细胞的分离和常规获得〜2×10 6</...

免疫系统激活发生在多个肾脏疾病和病理生理过程6,10,11,13。活跃​​的研究的潜在领域包括对免疫系统的激活的各种触发器，各种细胞类型涉及，在一个特定的疾病设置的细胞因子/趋化因子的模式，由特定的药物等。所有前述过程的调制为了举例说明，在缺血 - 再灌注损伤（急性肾损伤的模型），有增加的免疫细胞或骨髓衍生的造血细胞或CD45 +细胞在几个小时内，这是通过维修或纤维化的期间持续（6周后）5， 12。这些免疫细胞分泌促炎性和抗炎性细胞因子和趋化因子既来编排修复5,12的过程。目前，同时使用多个荧光团来标记细胞群体中的单个细胞悬浮液的能力已经与广告增加发泄现代流式细胞仪机与四到五激光器。这大大增加的能力来区分基于其功能状态3,7的细胞群。例如，为了精确地标记巨噬细胞为低 ，将在相同的样品体积是需要的栅极F4 / 80 低的CD11b 高 Ly6b 高 CD206至少3个荧光团为活细胞，CD45 +（白细胞）和Ly6G-（中性粒细胞）和这是与新的流式细胞仪3很可能的。然而，对于细胞因子的分泌，细胞增殖，细胞毒性，巨噬细胞活化和淋巴细胞和单核细胞的各种子集的数目的量化的下游分析不仅需要有良好的质量（活细胞，单峰），但细胞的足够数量。 在肾脏的免疫系统由两个适应性和先天组件和多种细胞类型1,7,13的。例如，在小鼠中两个小子分离（1.4×10 6个细胞）和它们的约5-15％（1,400-4,200）的总肾免疫细胞neys一起被报道含有2-17％（28,000-266,000） 的 CD45 +细胞是CD4 +细胞1 ，5,12。这些CD4 +细胞的一小部分（5-15％，70-630）是FOXP3 +细胞（ 图1）1。由于在细胞中的百分比这些步骤明智的减少，有时感兴趣的细胞群（在这种情况下，CD45 + CD4 +的FoxP3 +细胞）是由单纯的〜100个细胞表示。的CD45 + CD4 +的FoxP3 +细胞的数量少，因此必须有大量的总细胞分离并在细胞是质量好于下游的研究，例如细胞因子分泌测定。此外，它可能是必要的，因为亚群中不足够高的数字表示来执行量化测定以肾脏从2-3只小鼠相结合。因此，肾单核细胞群体的可靠和有效的隔离是可取的，以便螺柱y随肾脏疾病相关的免疫学光谱。 传统上，肾单核细胞的分离，研究人员使用了多种酶消化，如胶原酶1A或II包括DNA酶1 1,5,12的。它公知的是胶原酶具有与批号和由公司制造的不同而不同，因此有必要滴定为最佳浓度和温育4,14,15的持续时间的酶活性。此外，用胶原酶消化添加时间为切碎肾成小块，就必须肾脏条在EDTA中加热（37℃）浴和额外的时间为孵育的孵育终止反应。此外，较少的不育性可以针对某些下游程序需要的细胞培养来实现。更重要的是，根据所涉及的研究者和所有的变量，它会导致变异跨实验室数据和解释。近年来，随着机械组织干扰/均质16的发展，胶原酶消化步骤是完全可以避免和肾脏2的简单机械破碎代替。在此，我们展示了肾脏免疫细胞的日常免疫细胞提取隔离一个简单而有效的方法。重要的是，这种技术可以适用于其他柔软和非纤维组织如肝和脑16。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Stomacher 80 Biomaster lab system</td>
			<td>Seward</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stomacher 80 Classic bags</td>
			<td>Seward</td>
			<td>BA6040/STR</td>
			<td></td>
		</tr>
		<tr>
			<td>Sorvall Legend XFR Centrifuge</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td>Or equivalent equipment&#160;</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Electron Microscopy Sciences</td>
			<td>63514-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Analytical flow cytometer</td>
			<td>BD LSR-X20 Fortessa</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll&#160;</td>
			<td>Sigma</td>
			<td>P1644</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s phosphate buffered saline 1X (DPBS)</td>
			<td>Gibco, Life Technologies</td>
			<td>14190-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene tubes, no cap</td>
			<td>Becton Dickinson</td>
			<td>352002</td>
			<td></td>
		</tr>
		<tr>
			<td>Fixable Viability Stain</td>
			<td>BD Biosciences&#160;</td>
			<td>FVS510,&#160; 564406</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD16/32 (Clone: 93)</td>
			<td>EBioscience</td>
			<td>14-0161</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CD45 (clone: 30-F11)&#160; BV421&#160;</td>
			<td>BD Pharmingen</td>
			<td>103133/4</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Foxp3 (Clone: FJK-16s) APC</td>
			<td>EBioscience</td>
			<td>17-5773</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD127 (Clone: A7R34) PE/Cy7</td>
			<td>Biolegend</td>
			<td>135013/4</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CD44 (Clone: IM7) PerCP/Cy5.5</td>
			<td>Biolegend</td>
			<td>103031/2</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CD4 (Clone: RM4-5) APC-Cy7</td>
			<td>Biolegend</td>
			<td>100413/4</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CD8 (Clone: 53-6.7) BV785</td>
			<td>Biolegend</td>
			<td>100749/50</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Ly6G (Clone: 1A8) FITC</td>
			<td>Biolegend</td>
			<td>127605/6</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD11b (Clone: M1/70) PerCP-Cy5.5</td>
			<td>Biolegend</td>
			<td>101227/8</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-F4/80 (Clone: BM8) APC</td>
			<td>Biolegend</td>
			<td>123115/6</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD11c (Clone: N418) BV785</td>
			<td>Biolegend</td>
			<td>117335/6</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD301 (Clone: LOM-14) PE-Cy7</td>
			<td>Biolegend</td>
			<td>145705/6</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD26 (Clone: H194-112) PE</td>
			<td>Biolegend</td>
			<td>137803/4</td>
			<td></td>
		</tr>
		<tr>
			<td>100 &#956;m filter&#160;</td>
			<td>Fisher Scientific</td>
			<td>22363548</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand Tubes 50 ml</td>
			<td>Fisher</td>
			<td></td>
			<td>Or equivalent equipment&#160;</td>
		</tr>
		<tr>
			<td>Fisherbrand Tubes 15 ml</td>
			<td>Fisher</td>
			<td></td>
			<td>Or equivalent equipment&#160;</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Fisher chemical</td>
			<td>S5-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer pipette fine tip</td>
			<td>Samco Scientific</td>
			<td>232</td>
			<td>Or equivalent equipment&#160;</td>
		</tr>
		<tr>
			<td>Flow Cytometery Staining Buffer Solution</td>
			<td>EBioscience</td>
			<td>00-4222-26</td>
			<td>Or equivalent equipment&#160;</td>
		</tr>
		<tr>
			<td>1X RBC Lysis Buffer</td>
			<td>EBioscience</td>
			<td>00-4333-57</td>
			<td>Or equivalent equipment&#160;</td>
		</tr>
	</tbody>
</table>

reliable and high efficiency extraction of kidney immune cells

Immune system activation occurs in multiple kidney diseases and pathophysiological processes. The immune system consists of both adaptive and innate components and multiple cell types. Sometimes, the cell type of interest is present in very low numbers among the large numbers of total cells isolated from the kidney. Hence, reliable and efficient isolation of kidney mononuclear cell populations is important in order to study the immunological problems associated with kidney diseases. Traditionally, tissue isolation of kidney mononuclear cells have been performed via enzymatic digestions using different varieties and strengths of collagenases/DNAses yielding varying numbers of viable immune cells. Recently, with the development of the mechanical tissue disruptors for single cell isolation, the collagenase digestion step is avoided and replaced by a simple mechanical disruption of the kidneys after extraction from the mouse. Herein, we demonstrate a simple yet efficient method for the isolation of kidney mononuclear cells for every day immune cell extractions. We further demonstrate an example of subset analysis of immune cells in the kidney. Importantly, this technique can be adapted to other soft and non-fibrous tissues such as the liver and brain.

可运行的板的数量取决于该能够可靠地提取出了肾脏的免疫细胞的数目。这里，我们证明运行2板，一个用于T淋巴细胞，一个用于巨噬细胞/树突状细胞的能力。在T淋巴细胞面板，我们先来看看前向散射（FSC）和侧向散射（SSC）模式，描绘感兴趣的人群， 如图1（左上点图）。接着，生存力的标记，在这种情况下，一个可固定活力染色（FVS510）用于排除在分析?...

Watch this Scientific Journal Video about Reliable and High Efficiency Extraction of Kidney Immune Cells at JoVE.com

Reliable and High Efficiency Extraction of Kidney Immune Cells

Techniques that are reliable and efficient for the isolation of kidney immune cells are needed for downstream applications. This requires surface antibody labeling of a small number of kidney immune cells. Herein, we describe a concise method for isolation of kidney immune cells that seemingly achieves this goal.

肾脏免疫细胞的可靠和高效率的提取

Immune system activation occurs in multiple kidney diseases and pathophysiological processes.  The immune system consists of both adaptive and innate ...

reliable-and-high-efficiency-extraction-of-kidney-immune-cells

Research

JoVE Journal

Immunology and Infection

10.3K 次观看.  Department of Medicine, University of Missouri-Columbia and Harry S Truman Memorial Veteran's Hospital. Techniques that are reliable and efficient for the isolation of kidney immune cells are needed for downstream applications. This requires surface antibody labeling of a small number of kidney immune cells. Herein, we describe a concise method for isolation of kidney immune cells that seemingly achieves this goal.

视频: Reliable and High Efficiency Extraction of Kidney Immune Cells

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

在固定和活细胞，流式细胞仪测量钙蛋白酶活性

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

科研

免疫与感染

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

星状细胞的七个步骤

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

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 ...

Isolation of Functional Cardiac Immune Cells

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5 These immune cells, which include mast cells, T-cells and macrophages; store and release a variety of biologically active mediators including cytokines and proteases such as tryptase.6-8 These mediators have been shown to be key players in extracellular matrix metabolism by activating matrix metalloproteinases or causing collagen accumulation by modulating the cardiac fibroblasts' function.9-11 However, available techniques for isolating cardiac immune cells have been problematic because they use bacterial collagenase to digest the myocardial tissue. This technique causes activation of the immune cells and thus a loss of function. For example, cardiac mast cells become significantly less responsive to compounds that cause degranulation.12 Therefore, we developed a technique that allows for the isolation of functional cardiac immune cells which would lead to a better understanding of the role of these cells in cardiac disease.13, 14
This method requires a familiarity with the anatomical location of the rat's xiphoid process, axilla and falciform ligament, and pericardium of the heart. These landmarks are important to increase success of the procedure and to ensure a higher yield of cardiac immune cells. These isolated cardiac immune cells can then be used for characterization of functionality, phenotype, maturity, and co-culture experiments with other cardiac cells to gain a better understanding of their interactions.

This method for isolating functional immune cells from the heart provides an alternative to the conventional methods of collagenase digestion, which causes unwanted immune cell activation, resulting in a decreased responsiveness of these cells. Our method of isolation yields functional cardiac immune cells by avoiding problems associated with enzymatic digestion.

心脏功能免疫细胞的分离

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5  These immune cells, which ...

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.
Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.
Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KIgfp/gfp mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.

活体成像的鼠标使用2 - 光子显微镜的胸腺

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

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 of Double Negative &#945;&#946; T Cells from the Kidney

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in various immune responses. DN T cells are a unique immune cell type that has been implicated in regulating immune and autoimmune responses and tolerance to allotransplants1-6. DN T cells are, however, rare in peripheral blood and secondary lymphoid organs (spleen and lymph nodes), but are major residents of the normal kidney. Very little is known about their pathophysiologic function7 due to their paucity in the periphery. We recently described a comprehensive phenotypic and functional analysis of this population in the kidney8 in steady state and during ischemia reperfusion injury. Analysis of DN T cell function will be greatly enhanced by developing a protocol for their isolation from the kidney.
Here, we describe a novel protocol that allows isolation of highly pure ab CD4+ CD8+ T cells and DN T cells from the murine kidney. Briefly, we digest kidney tissue using collagenase and isolate kidney mononuclear cells (KMNC) by density gradient. This is followed by two steps to enrich hematopoietic T cells from 3% to 70% from KMNC. The first step consists of a positive selection of hematopoietic cells using a CD45+ isolation kit. In the second step, DN T cells are negatively isolated by removal of non-desired cells using CD4, CD8, and MHC class II monoclonal antibodies and CD1d &#945;-galcer tetramer. This strategy leads to a population of more than 90% pure DN T cells. Surface staining with the above mentioned antibodies followed by FACs analysis is used to confirm purity.

DNabT cells are rare among peripheral T cells; however, they are abundant in certain non-lymphoid tissues. Difficulty of isolating DN T cells from non-lymphoid tissue hinders their functional analysis despite increasing recognized pathophysiologic significance. We describe a novel protocol for isolation of highly purified DN T cells from murine kidney.

从肾脏双重否定αβ性T细胞的分离

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

葡聚糖增强小鼠和人原发性 NK 细胞的慢转导效率

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

Isolation and Identification of Extravascular Immune Cells of the Heart

The immune system is an essential component of a healthy heart. The myocardium is home to a rich population of different immune cell subsets with functional compartmentalization both during steady state and during different forms of inflammation. Until recently, the study of immune cells in the heart required the use of microscopy or poorly developed digestion protocols, which provided enough sensitivity during severe inflammation but were unable to confidently identify small &#8212; but key &#8212; populations of cells during steady state. Here, we discuss a simple method combining enzymatic (collagenase, hyaluronidase and DNAse) and mechanical digestion of murine hearts preceded by intravascular administration of fluorescently-labelled antibodies to differentiate small but unavoidable intravascular cell contaminants. This method generates a suspension of isolated viable cells that can be analyzed by flow cytometry for identification, phenotyping and quantification, or further purified with fluorescence-activated cell sorting or magnetic bead separation for transcriptional analysis or in vitro studies. We include an example of a step-by-step flow cytometric analysis to differentiate the key macrophage and dendritic cell populations of the heart. For a medium sized experiment (10 hearts) the completion of the procedure requires 2&#8211;3 h.

This protocol presents a simple and efficient method to isolate, identify and quantify immune cells residing in the myocardium of mice during steady state or inflammation. The protocol combines enzymatic and mechanical digestion for the generation of a single cell suspension that can be further analyzed by flow cytometry.

心脏外免疫细胞的分离与鉴定

The immune system is an essential component of a healthy heart. The myocardium is home to a rich population of different immune cell subsets with ...

High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In response to these damages, complex molecular machineries rapidly reseal the plasma membrane to restore its barrier function and maintain cell survival. Despite 60 years of research in this field, we still lack a thorough understanding of the cell resealing machinery. With the goal of identifying cellular components that control plasma membrane resealing or drugs that can improve resealing, we have developed a fluorescence-based high-throughput assay that measures the plasma membrane resealing efficiency in mammalian cells cultured in microplates. As a model system for plasma membrane damage, cells are exposed to the bacterial pore-forming toxin listeriolysin O (LLO), which forms large 30-50 nm diameter proteinaceous pores in cholesterol-containing membranes. The use of a temperature-controlled multi-mode microplate reader allows for rapid and sensitive spectrofluorometric measurements in combination with brightfield and fluorescence microscopy imaging of living cells. Kinetic analysis of the fluorescence intensity emitted by a membrane impermeant nucleic acid-binding fluorochrome reflects the extent of membrane wounding and resealing at the cell population level, allowing for the calculation of the cell resealing efficiency. Fluorescence microscopy imaging allows for the enumeration of cells, which constitutively express a fluorescent chimera of the nuclear protein histone 2B, in each well of the microplate to account for potential variations in their number and allows for eventual identification of distinct cell populations. This high-throughput assay is a powerful tool expected to expand our understanding of membrane repair mechanisms via screening for host genes or exogenously added compounds that control plasma membrane resealing.

Here we describe a high-throughput fluorescence-based assay that measures the plasma membrane resealing efficiency through fluorometric and imaging analyses in living cells. This assay can be used for screening drugs or target genes that regulate plasma membrane resealing in mammalian cells.

哺乳动物细胞血浆膜检测效率的高通量测量

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In ...