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>

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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><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&amp;nbsp;</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&amp;nbsp;</td><td>Sigma</td><td>P1644</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;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&amp;nbsp;</td><td>FVS510,&amp;nbsp; 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)&amp;nbsp; BV421&amp;nbsp;</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 &amp;mu;m filter&amp;nbsp;</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&amp;nbsp;</td></tr><tr><td>Fisherbrand Tubes 15 ml</td><td>Fisher</td><td></td><td>Or equivalent equipment&amp;nbsp;</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&amp;nbsp;</td></tr><tr><td>Flow Cytometery Staining Buffer Solution</td><td>EBioscience</td><td>00-4222-26</td><td>Or equivalent equipment&amp;nbsp;</td></tr><tr><td>1x RBC Lysis Buffer</td><td>EBioscience</td><td>00-4333-57</td><td>Or equivalent equipment&amp;nbsp;</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

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 ...

科研

免疫与感染

Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model substrates for organ-scale tissue development. Sprague Dawley rat kidneys are cannulated by inserting a catheter into the renal artery and perfused with a series of low-concentration detergents (Triton X-100 and sodium dodecyl sulfate (SDS)) over 26 hr to derive intact, whole-kidney scaffolds with intact perfusable vasculature, glomeruli, and renal tubules. Following decellularization, the renal scaffold is placed inside a custom-designed perfusion bioreactor vessel, and the catheterized renal artery is connected to a perfusion circuit consisting of: a peristaltic pump; tubing; and optional probes for pH, dissolved oxygen, and pressure. After sterilizing the scaffold with peracetic acid and ethanol, and balancing the pH (7.4), the kidney scaffold is prepared for seeding via perfusion of culture medium within a large-capacity incubator maintained at 37 &#176;C and 5% CO2. Forty million renal cortical tubular epithelial (RCTE) cells are injected through the renal artery, and rapidly perfused through the scaffold under high flow (25 ml/min) and pressure (~230 mmHg) for 15 min before reducing the flow to a physiological rate (4 ml/min). RCTE cells primarily populate the tubular ECM niche within the renal cortex, proliferate, and form tubular epithelial structures over seven days of perfusion culture. A 44 &#181;M resazurin solution in culture medium is perfused through the kidney for 1 hr during medium exchanges to provide a fluorometric, redox-based metabolic assessment of cell viability and proliferation during tubulogenesis. The kidney perfusion bioreactor permits non-invasive sampling of medium for biochemical assessment, and multiple inlet ports allow alternative retrograde seeding through the renal vein or ureter. These protocols can be used to recellularize kidney scaffolds with a variety of cell types, including vascular endothelial, tubular epithelial, and stromal fibroblasts, for rapid evaluation within this system.

This protocol describes decellularization of Sprague Dawley rat kidneys by antegrade perfusion of detergents through the vasculature, producing acellular renal extracellular matrices that serve as templates for repopulation with human renal epithelial cells. Recellularization and use of the resazurin perfusion assay to monitor growth is performed within specially-designed perfusion bioreactors.

上皮细胞复育和肾组织的发展准备啮齿动物细胞外基质的支架

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model ...

生物工程

Rapid Depletion of Renal Macrophages using Human CD59/Intermedilysin Cell Ablation Tool

Renal macrophages (RMs) are essential for kidney health, orchestrating immune surveillance, tissue homeostasis, and responses to injury. Previously, we reported the use of a human CD59 (hCD59)/intermedilysin (ILY) cell ablation tool to study the distinct fate, dynamics, and niches of RMs of bone marrow or embryonic origin. RMs originate from yolk sac-derived macrophages, fetal liver monocytes, and bone marrow-derived monocytes and are maintained in adulthood through local proliferation and recruitment of circulating monocytes. Here, we report a detailed protocol for the selective ablation of RMs to study their regeneration, including 1) generation and characterization of the Cre-inducible expression of hCD59 in mouse RMs, 2) purification of ILY and characterization of ILY activity, 3) induction of hCD59 expression on RMs in compound mice, and 4) characterization of regeneration after ILY-mediated RM ablation. ILY specifically and rapidly depletes RMs in compound mice, with efficient macrophage ablation within 1 day of ILY administration. Renal macrophage regeneration began by day 3 post-ablation, with ~88% recovery by day 7. This model offers a powerful tool for studying macrophage biology and can be used for selectively ablating other cell populations in the kidney, liver, and fatty tissues to investigate their function and regeneration.

A protocol is reported here for the selective ablation of renal macrophages to study their regeneration using the human CD59/intermedilysin cell ablation tool. This method is also applicable for studying the function and regeneration of the other cell populations in the kidney, liver, and fatty tissue.

使用人 CD59/中间溶血素细胞消融工具快速耗竭肾巨噬细胞

Renal macrophages (RMs) are essential for kidney health, orchestrating immune surveillance, tissue homeostasis, and responses to injury. Previously, we ...

发育生物学

A Novel Clinical Grade Isolation Method for Human Kidney Perivascular Stromal Cells

Mesenchymal Stromal Cells (MSCs) are tissue homeostatic and immune modulatory cells that have shown beneficial effects in kidney diseases and transplantation. Perivascular Stromal Cells (PSCs) share characteristics with bone marrow MSCs (bmMSCs). However, they also possess, most likely due to local imprinting, tissue-specific properties and play a role in local tissue homeostasis. This tissue specificity may result in tissue specific repair, also within the human kidney. We previously showed that human kidney PSCs (kPSCs) have enhanced kidney epithelial wound healing whereas bmMSCs did not have this potential. Moreover, kPSCs can ameliorate kidney injury in vivo. Therefore, kPSCs constitute an interesting source for cell therapy, particularly for kidney diseases and renal transplantation. Here we show the detailed isolation and culture method for kPSCs from transplant-grade human kidneys based on whole-organ perfusion of digestive enzymes via the renal artery and enrichment for the perivascular marker NG2. In this way, large cell quantities can be obtained that are suitable for cellular therapy.

Here we present a novel clinical grade isolation and culture method for kidney Perivascular Stromal Cells (kPSCs) based on whole organ perfusion with digestive enzymes and NG2-cell enrichment. With this method, it is possible to acquire sufficient cell numbers for cellular therapy.

人类肾脏血管周围间质细胞新型临床分级分离方法

Mesenchymal Stromal Cells (MSCs) are tissue homeostatic and immune modulatory cells that have shown beneficial effects in kidney diseases and ...

医学

Detection of MicroRNA Expression in the Kidneys of Immunoglobulin A Nephropathic Mice

Immunoglobulin A (IgA) nephropathy is a type of primary glomerulonephritis characterized by the abnormal deposition of IgA, leading to the end-stage renal failure. In recent years, the involvement of microRNAs (miRNAs) has been reported in the pathogenesis of IgA nephropathy. However, there is no established method for profiling miRNAs in IgA nephropathy using small animal models. Therefore, we developed a reliable method for analyzing miRNA in the kidney of an IgA mouse model (HIGA mouse). The goal of this protocol is to detect the altered expression levels of miRNAs in the kidneys of HIGA mice when compared with the levels in kidneys of control mice. In brief, this method consists of four steps: 1) obtaining kidney samples from HIGA mice; 2) purifying total RNA from kidney samples; 3) synthesizing complementary DNA from total RNA; and 4) quantitative reverse transcription polymerase chain reaction (qRT-PCR) of miRNAs. Using this method, we successfully detected the expression levels of several miRNAs (miR-155-5p, miR-146a-5p, and miR-21-5p) in the kidneys of HIGA mice. This new method can be applied to other studies profiling miRNAs in IgA nephropathy.

microRNAs are involved in the pathogenesis of IgA nephropathy. We have developed a reliable method for detecting microRNA expression levels in the kidneys of an IgA nephropathy mouse model (HIGA mice). This new method will facilitate to check for miRNAs involvement in IgA nephropathy.

检测免疫球蛋白A肾病小鼠肾脏中的microRNA表达

Immunoglobulin A (IgA) nephropathy is a type of primary glomerulonephritis characterized by the abnormal deposition of IgA, leading to the end-stage renal ...

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 ...

An Efficient Sieving Method to Isolate Intact Glomeruli from Adult Rat Kidney

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by proteinuria which can eventually lead to chronic and end-stage renal disease. The glomerulus is a complex apparatus responsible for the filtration of plasma from the body. In disease, structural integrity is lost and allows for the abnormal leakage of plasma contents into the urine. A method to isolate and examine glomeruli in culture is critical for the study of these diseases. In this protocol, an efficient method of retrieving intact glomeruli from adult rat kidneys while conserving structural and morphological characteristics is described. This process is capable of generating high yields of glomeruli per kidney with minimal contamination from other nephron segments. With these glomeruli, injury conditions can be mimicked by incubating them with a variety of chemical toxins, including protamine sulfate, which causes foot process effacement and proteinuria in animal models. Degree of injury can be assessed using transmission electron microscopy, immunofluorescence staining, and western blotting. Nephrin and Wilms Tumor 1 (WT1) levels can also be assessed from these cultures. Due to the ease and flexibility of this protocol, the isolated glomeruli can be utilized as described or in a way that best suits the needs of the researcher to help better study glomerular health and structure in diseased states.

The main focus of this protocol is to efficiently isolate viable primary glomeruli cultures with minimal contaminants for use in a variety of downstream applications. The isolated glomeruli retain structural relationships between component cell types and can be cultured ex vivo for a short time.

一种从成年大鼠肾脏中分离完整型肾小球的有效筛分方法

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by ...

Optical Clearing and Imaging of Immunolabeled Kidney Tissue

Optical clearing techniques render tissue transparent by equilibrating the refractive index throughout a sample for subsequent three-dimensional (3-D) imaging. They have received great attention in all research areas for the potential to analyze microscopic multicellular structures that extend over macroscopic distances. Given that kidney tubules, vasculature, nerves, and glomeruli extend in many directions, which have been only partially captured by traditional two-dimensional techniques so far, tissue clearing also opened up many new areas of kidney research. The list of optical clearing methods is rapidly growing, but it remains difficult for beginners in this field to choose the best method for a given research question. Provided here is a simple method that combines antibody labeling of thick mouse kidney slices; optical clearing with cheap, non-toxic and ready-to-use chemical ethyl cinnamate; and confocal imaging. This protocol describes how to perfuse kidneys and use an antigen-retrieval step to increase antibody- binding without requiring specialized equipment. Its application is presented in imaging different multicellular structures within the kidney, and how to troubleshoot poor antibody penetration into tissue is addressed. We also discuss the potential difficulties of imaging endogenous fluorophores and acquiring very large samples and how to overcome them. This simple protocol provides an easy-to-setup and comprehensive tool to study tissue in three dimensions.

The combination of antibody labeling, optical clearing, and advanced light microscopy allows three-dimensional analysis of complete structures or organs. Described here is a simple method to combine immunolabeling of thick kidney slices, optical clearing with ethyl cinnamate, and confocal imaging that enables visualization and quantification of three-dimensional kidney structures.

免疫标记肾组织的光学清除与成像

Optical clearing techniques render tissue transparent by equilibrating the refractive index throughout a sample for subsequent three-dimensional (3-D) ...

Nuclei Isolation from Adult Mouse Kidney for Single-Nucleus RNA-Sequencing

The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are executed by multiple cell types arranged in a complex architecture across the corticomedullary axis of the organ. Recent advances in single-cell transcriptomics have accelerated the understanding of cell type-specific gene expression in renal physiology and disease. However, enzyme-based tissue dissociation protocols, which are frequently utilized for single-cell RNA-sequencing (scRNA-seq), require mostly fresh (non-archived) tissue, introduce transcriptional stress responses, and favor the selection of abundant cell types of the kidney cortex resulting in an underrepresentation of cells of the medulla.
Here, we present a protocol that avoids these problems. The protocol is based on nuclei isolation at 4 &#176;C from frozen kidney tissue. Nuclei are isolated from a central piece of the mouse kidney comprised of the cortex, outer medulla, and inner medulla. This reduces the overrepresentation of cortical cells typical for whole-kidney samples for the benefit of medullary cells such that data will represent the entire corticomedullary axis at sufficient abundance. The protocol is simple, rapid, and adaptable and provides a step towards the standardization of single-nuclei transcriptomics in kidney research.

Here, we present a protocol to isolate high-quality nuclei from frozen mouse kidneys that improve the representation of medullary kidney cell types and avoids the gene expression artifacts from enzymatic tissue dissociation.

从成年小鼠肾脏中分离细胞核以进行单核RNA测序

The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are ...

生物学

Optimizing Isolation and Purification of Murine Glomerular Mesangial Cells

Mesangial cells (MCs) are stromal cells located in the middle space of the glomerulus, with pivotal functions in glomerular homeostasis. Methods for isolating, purifying, and culturing glomerular MCs have been developed and optimized since the 1980s for use in biomedical research, particularly in the field of nephrology. Mice are the most frequently used experimental animal models in research on renal diseases. In this study, we developed an optimized protocol for murine MCs isolation and ex vivo cell culture. These cells can be passaged multiple times, frozen, revived, and cultured without compromising cell growth or protein expression. This optimized approach significantly reduces the study duration for researchers and enables long-term cell preservation. The necessary equipment for the procedures is easily accessible in a basic biomedical laboratory, and the procedural steps are straightforward. The acquisition of target cells requires only 2-3 weeks, a reduction of at least 1 week compared to existing methods.

This study developed an optimized protocol for the isolation of murine mesangial cells (MCs) and their ex vivo cell culture. These cells can be passaged multiple times, frozen, revived, and cultured without compromising cell growth or protein expression.

优化小鼠肾小球系膜细胞的分离和纯化

Mesangial cells (MCs) are stromal cells located in the middle space of the glomerulus, with pivotal functions in glomerular homeostasis. Methods for ...