笔者想感谢圭多范霍文和鲁Deboel，鲁汶，谁是在吉森的迈进实验室建立初级睾丸支持细胞的分离极有帮助。莫妮卡Fijak，吉森，承认她与图1A和咨询帮助。研究由德意志研究联合会授予通过BH 93 / 1-1（SB）和资助国际研究研究生院吉林大学吉森（德国）/莫纳什大学（澳大利亚墨尔本）1871年GRK（SB）的支持。从黑森州州的赠款被称为"MännlicheInfertilität贝Infektion＆Entzündung"（MIBIE）节目"兰德斯进攻性楚发展协会Wissenschaftlich-ökonomischerExzellenz"（LOEWE）中也获得支持。

1.动物伦理声明这里描述的实验按照Regierungspräsidium德国吉森准则执行，因为地方当局并确认实践照顾和动物用于实验目的德语代码（许可号:GI 20/23上午十时正A 31 / 2012）。 2.媒体，酶溶液和动物的制备<ol><li>由1克碘溶在100ml乙醇中制备1％碘醇。 </li><li>制备2×500毫升Dulbecco&#39;s磷酸盐缓冲盐水（PBS）不含Ca 2+和Mg 2+ 7.5毫升D-葡萄糖（100克/升）和5毫升100×青霉素/链霉素原液（15毫克/毫升ð补充各葡萄糖，50 U / ml的青霉素和50μg/ ml的链霉素最终）。 </li><li>补充1×500毫升罗斯韦尔园区纪念研究所（RPMI）用5ml 100×青霉素/链霉素原液（50单位/毫升青霉素和50微克/毫升链霉素鳍1640培养基人）。 </li><li>制备胰蛋白酶DNA酶I溶液（2.5毫克/毫升胰蛋白酶和10微克/毫升DNA酶I）加入25毫克胰蛋白酶和0.1毫升DNA酶I（1毫克/毫升DNA酶I）至9.9毫升PBS中。 </li><li>溶解在5ml PBS中的胰蛋白酶50mg的胰蛋白酶抑制剂的抑制剂溶液（10毫克/毫升）。溶解25mg的胰蛋白酶抑制剂在10毫升PBS中的胰蛋白酶抑制剂B液（2.5毫克/毫升）。 </li><li>对于胶原酶透明质酸酶 - 脱氧核糖​​核酸酶I（DNA酶I）溶液溶解10毫克胶原酶A（1毫克/毫升终浓度），10毫克透明质酸酶（1mg / ml的终浓度）和0.1毫升DNA酶I（1毫克/毫升DNA酶I）（ 10微克，10毫升的PBS / ml的终浓度）。 </li><li>通过加入10毫克透明质酸酶（1mg / ml的终浓度）和0.1毫升DNA酶I溶液（1毫克/毫升）（10微克/毫升终浓度）至9.9毫升的PBS制备透明质酸酶DNA酶I溶液。 </li><li>准时交货Wistar大鼠，使得它们在塞尔托利细胞分离的日龄19天。 注意:所述的协议被设计为10只，但是可以修改为至少5和最多20只大鼠。所有溶液的体积必须作相应的调整。 </li><li>通过在100毫升异丙醇溶解0.35克油红O制备油红O原液。搅拌O / N，通过0.2微米和储存在室温为长达一年的筛选。 </li><li>通过混合6毫升油红O贮存液用4ml水制备油红O染色溶液（3:2）。通过后0.2微米的10-20分钟的过滤器。接下来的2小时内使用。 </li><li>制备4％（重量/体积）通过加入4g多聚甲醛粉末（PFA）向80ml 1×PBS中的搅拌的同时轻轻地在通风橱甲醛溶液。热到〜60℃，加入1毫升的1摩尔的NaOH，并继续搅拌，直到多聚甲醛溶解。让溶液冷却至室温，调整用1M HCl将pH至7.4并定容至100毫升，1×PBS中。过滤溶液（0.45微米），并使用在等分新鲜或储存在-20℃下几个月。 注:聚合甲醛（PFA）解聚至单体甲醛水。这个过程是通过适度加热和弱碱性pH催化。刚准备所有的酶溶液和过滤灭菌（0.2μM）他们在使用当天。如果一个不同的制造商的酶（ 参见材料/设备 表 ）时，小心地调整其浓度/活性。 在下面的协议的"吸管"代表血清吸管。 </li></ol> 3.曲细精管的制备<ol><li>麻醉10只雄性Wistar大鼠逐一在使用CO 2的干燥器，并且取代每分钟腔室体积的至少20％的流速。通过挤压一脚垫等到呼吸停止，测试麻醉，并且如果不存在反应通过颈脱位安乐死每只大鼠。流水下切片颈静脉和颈动脉（阴道carotica）沥血。通过用70％乙醇擦拭消毒腹部。 </li><li>使用镊子解除从腹部亩皮肤克莱斯和切出一个从耻骨联合到胸骨（图2A）延伸的椭圆形皮肤瓣，向上拍打它放到胸前。接下来，抢腹肌，并从耻骨联合到胸骨使内侧切口。 <ol><li>挤压与骨盆拇指腹部，以推动睾丸从下骨盆向上。挑附睾脂肪垫（图2B）用钳子用于进一步拉起睾丸。切断精索（图2B），留下白膜完整，并收集所有的睾丸在20毫升PBS在50毫升的锥形管（图2C）。 </li></ol></li><li>收集所有睾丸后，通过添加1％的等体积（20毫升）（重量/体积）的乙醇碘消毒他们。倒置该管的两倍，并立即倒出上清液。迅速用25ml的PBS每个（图2D）洗睾丸两次。 </li><li>转移睾丸至培养皿含有ING15毫升PBS（图2E）。一端牢牢把握每一个睾丸被镊子，做一个小切口进入在另一端的白膜，并挤掉使用封闭的剪刀片作为一种工具刮肾小管。 注意:肾小管仍应形成紧凑睾丸形束，只有少数小管延伸到所述溶液中，以使均匀的酶促消化（图2F）。 </li><li>传输解封装睾丸含有10ml胰蛋白酶DNA酶I溶液的100毫升螺旋盖瓶中。在32℃（120振荡/分钟）执行在振荡水浴中消化。 4分钟后，评价与小管的分散肉眼肾小管。一旦管开始分散到溶液中，立即停止消化。如果需要的话，继续孵育达2分钟。 </li><li>通过加入5ml胰蛋白酶抑制剂溶液A停止胰酶消化吹打起来，充分搅拌溶解上下3-4时间S使用10毫升吸管，并将其转移到50毫升锥形管中。 </li><li>孵育5分钟后观察小管通过单位重力沉淀，仔细用10毫升吸管从步骤3.6去除上清，加入10毫升胰蛋白酶抑制剂B液，以全面停止胰酶消化。通过上下吹打2-3次，重悬管。 </li><li>为了除去污染质细胞洗管9次用25毫升的PBS每个。用25毫升吸管重悬在每洗管，让他们通过单位重力10分钟解决。始终使用相同的25毫升吸管移液PBS，再悬浮和除去上清液。 </li></ol>管周细胞4.移动/隔离（PTC）的<ol><li>传输所有小管到100毫升螺旋盖瓶中，并添加胶原酶透明质酸酶DNA酶I溶液（图3A）。在32℃在振荡水浴中10分钟进行消化（120振荡/分钟）。 </li><li>吸管悬浮液在载玻片上的下降，快速检查的倒置光学显微镜的常规细胞培养下的小管在放大100倍。如果消化不够先进持续增加2分钟孵化（不计所需显微镜检查的时间）。 注意:在此步骤期间所有管获得缩短的长度和细管边缘应该变得粗糙（图3B 和 C）。粗糙的边缘是指示为管周细胞的释放。 </li><li>用25毫升吸管从步骤3.8与消化小管转移悬浮液到50毫升锥形管中。用10ml PBS洗100毫升瓶装和它在锥形管添加到管。允许消化小管到由单位重力10分钟沉降，小心地吸出上清液，将含有的PTC，再次使用25毫升吸管，并将其转移到一个新的锥形管中。在过去的几毫升的愿望使用奥德5毫升吸管R以防止管的任何结转。 </li><li>添加20毫升RPMI补充有10％热灭活的胎牛血清（FBS）的1640培养基中收集到的小管的上清液，在300×g离心在RT 10分钟不使用断混合和离心。 </li><li>小心倒出上清液并在20ml重悬丸状的PTC的RPMI在含有每行16毫升培养基5 T75烧瓶补充有10％热灭活FBS 1640培养基和种子各4mL。在5％CO 2气氛中 （图4A）在37℃。 </li><li>在培养的第 3天trypsinize PTC和分裂他们1:在含有补充有10％热灭活FBS16毫升RPMI1640培养基的T75烧瓶2各自（产率共10瓶）（图4B）。在5％CO 2气氛中于37℃继续温育。 </li><li>在培养的第 5天再次trypsinize PTC和板他们上取决于实验6孔或24孔板（每板1 T75瓶）。实验可以在第6天进行。 </li></ol> 5.隔离支持细胞（SCS） <ol><li>用25毫升吸管悬浮步骤4.3彻底在25毫升PBS的解决管，让他们通过单位重力12分钟解决。使用相同的25毫升吸管移液PBS，小管和去除上清液再悬浮。重复洗3次（4次洗涤总共）。 </li><li>最后一次洗涤消化小管转移至另外100之后ml旋盖含透明质酸酶的DNase I溶液瓶。在32℃（120振荡/分钟）执行在振荡水浴中消化。 </li><li> 5分钟后再次检查管通过放大100倍的光学显微镜检查，如步骤4.2所述。 注:短管，管聚集和释放的细胞应该是可见的（图3D）。 <ol><li>允许管状聚集体沉降10分钟，吸出上清液我们ING 25毫升吸管。洗涤聚合用25毫升的PBS和10分钟的每个洗涤步骤（图3E）沉降时间4倍。 </li></ol></li><li>添加20毫升的RPMI 1640培养基中，并通过装在有20毫升注射器18G的针通过该悬浮液的10倍。避免气泡产生。 注意:拉动悬浮液中并通过针头被认为是一个道次。细胞通过流体动力剪切破坏和均化是在倾向于聚集细胞的制备的标准技术。细胞的穿过小内径的皮下注射针是不太可能对他们的功能特性产生影响25。 图5C 和 D示出了纯化的支持细胞在培养第4天的正常超微结构。 <ol><li>吸管悬浮液在载玻片上并快速地检查一个倒置光学显微镜的常规细胞培养下的小管在10倍的放大倍率，如果AGG一滴regates都分散（图3F）。过滤通过70微米的细胞过滤细胞悬液，以获得在滤液纯单细胞悬液。 </li></ol></li><li>离心操作5.4在200 XG在室温下10分钟的滤液不使用休息。用25毫升吸管小心吸出上清液，并悬浮支持细胞在40ml的RPMI 1640培养基（无血清）。 </li><li>混合100μl的细胞悬浮液用100μl台盼蓝染色，并用一个纽鲍尔改进腔室计数细胞。根据该计数结果调整细胞浓度为3×10 6个细胞/ ml。种子1毫升，每孔在6孔板（= 1天）。如果油红O染色（第6步）计划把盖玻片到一个或播种支持细胞前的几个井。 注意:在悬浮Sertoli细胞迅速沉降使得管应的小区等分试样采取用吸管之前进行简单振摇每一次。直到4他们紧紧ATT天ACH到底层。 </li><li>为了除去在第4天的浮动或粘附生殖细胞（图4C）洗涤用于电镀孤立塞尔托利细胞（步骤5.6），用PBS（图4D）的6孔板的每个孔中。吸出介质，加入1-2毫升的PBS至各孔中，水平摇上剧烈的表，但没有溢出PBS中的6孔板。重复洗涤3次，并在第5天及6（图4E）执行相同的洗涤程序。 注意:在第4天塞尔托利细胞已牢固地连接并显示像倒置光学显微镜（图4C）根据图案的鹅卵石。实验可以从起7天来执行。 <ol><li> （替代对步骤5.7）为免除的浮动和粘附生殖细胞接种于6孔板之后执行低渗休克3天。取下中，加入1ml 20毫米的Tris pH值7.5，直接取出冰箱里的。吸出Tris缓冲液后90〜120秒，但不后来。洗涤细胞两次，用PBS，并加2ml的RPMI 1640培养基（无血清）中。任选地，进行实验的第二天。 注意:低渗休克的较长的持续时间越生殖细胞可被删除，但更Sertoli细胞迷路以及。所述第一PBS洗涤应该为了不取代任何支持细胞很快但小心进行。后直接不同尺寸的低渗处理大量液泡可以在相差显微镜细胞质中观察到。液泡消失低渗处理后的几个小时内。 </li></ol></li></ol> 6，油红O染色<ol><li>在执行RT的所有步骤。第7天洗支持细胞生长在盖玻片（步骤5.6），用PBS两次，用PBS 10％福尔马林固定。 10分钟后代替甲醛与新鲜溶液，并继续固定另一个60分钟。 注意:所有解决方案都加入到孔中（多个）用盖玻片下一步骤之前吸出。 </l我&gt; <li>吸甲醛，用清水冲洗细胞两次，并添加60％的异丙醇。 5分钟后，使细胞在空气中干燥几分钟。 </li><li>加入每孔1毫升油红O染色解决方案，并孵育10分钟。吸出溶液，并用水洗涤细胞立即4次和挂接盖玻片在甘油的PBS（9:1）。采取与常规光学显微镜（图4F）亮场图像。 </li></ol> 7.免疫荧光染色<ol><li> （来自步骤4.5）或生长在用PBS盖玻片两次塞尔托利细胞（来自步骤5.5）洗涤PTC和用4％PFA固定它们在室温10分钟。 </li><li>用在PBS中5％BSA在RT 1小时孵育。丢弃BSA-PBS溶液，并添加含有肌动蛋白（平滑肌）新鲜的BSA-PBS溶液（对PTC）的或波形蛋白（用于支持细胞）初级抗体（稀释1:100为两种抗体）并在4℃CO / N孵育。 注意:孵化用BSA块非特异性的第一抗体的结合。 </li><li>洗涤盖玻片5分钟3次在PBS-0.1％吐温20和绿色荧光染料缀合的山羊抗小鼠二抗（稀释1:1000）孵育在室温下在黑暗中1小时。 </li><li>洗涤盖玻片5分钟3次在PBS-0.1％吐温20和DAPI安装介质装入它们。 </li></ol> 8.超微结构分析<ol><li>生长在用PBS将6cm细胞培养板洗涤塞尔托利细胞（来自步骤5.6）和在2.5％戊二醛，2.5％多聚甲醛和0.05％苦味酸在67毫二甲砷酸缓冲液中的混合物修复它们为2小时，在4℃（根据伊藤和Karnovsky 26 pH为7.4）。 </li><li>在1％的四氧化锇随后的O / N培养在0.3％乙酸双氧铀溶于50mM马来酸盐缓冲液（pH 5）执行后固定。嵌入样品中根据标准程序嵌入媒体。切薄片，用柠檬酸铅对比它们并用电子显微镜检查。 </li></ol>

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Biophys. Acta. 1722, 6-14 (2005).</li></ol>

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The seminiferous tubules are bounded by a circular layer of peritubular cells and a basal lamina on the luminal side. Sertoli cells are resting on the basal lamina, establish the blood testis barrier through formation of occluding junctions between adjacent cells and provide the structural framework for the organisation of the seminiferous epithelium. Sertoli cells and adjacent spermatogenic cells maintain intimate contacts throughout germ cell development providing physical contact and communication alike. Therefore, wh...

睾丸产生雄配子和性激素， 即雄激素。器官是由两个车厢。在间质车厢，代表总睾丸容积1的约10-12％，类固醇取睾丸间质细胞内进行。管状隔室表示睾丸体积1的约60-80％，并含有生殖细胞和两种类型的体细胞-管周细胞和支持细胞。睾丸是由结缔组织隔膜分成小叶250-300，每个都包含1-3高度错综复杂的曲细精管。这些细管是由基底膜，胶原片，和管周细胞的周层（图1A）包围。 生殖上皮位于基底膜的腔侧。支持细胞是跨越从基底膜管腔整个生殖上皮大细长细胞。他们强烈ATT痛的基底膜，形成通过基底组织紧密连接，从间质闭塞生发上皮和表示血睾屏障的连续的蜂窝片。支持细胞有显着的细胞质预测和后果，使他们能够进入与种属特异性，但固定数量的生殖细胞紧密形态和功能联系。二倍体胚干细胞增殖和分化成精原细胞。 在减数分裂我短暂的四倍体精母细胞产生减数分裂II期间进一步分为四个单倍体精子细胞发展。所有生殖细胞通过细胞质桥，使得它们形成一个蜂窝网互连。精子细胞的成熟过程中的主要事件是细胞质的大部分挤出，形成残体，在一个叫做精子的过程。残体被支持细胞吞噬。然后晚期精子细胞被释放到管状内腔和输送到附睾进一步成熟。支持细胞和生殖细胞似乎相互配合地形精子发生和功能。 个体睾丸细胞类型的启动准备近一个世纪前，当小睾丸片进行培养和细胞类型鉴定显微镜2。通过打开用细尖镊子白膜后肾小管小心剥离它以后可能分离肾小管及间质车厢3。在1975年威尔士和Wiebe的，以便从附着间质组织和胰酶处理以除去外管周细胞层 4的释放管引入一个胶原酶处理。从早期年轻幼鼠（约20天）就在其中所使用的支持细胞构成，因为精子还没有开始的肾小管上皮细胞人口的很大一部分。在这个年龄段大鼠塞尔特OLI细胞停止分裂，和相邻小区之间的紧密连接形成，使血睾屏障建立5。 独立的威尔士和韦博Dorrington 等人介绍的胰蛋白酶结合，并脱氧核糖核酸其次soybeen胰蛋白酶抑制剂和胶原酶的治疗，发表于同年6。两组以便产生用于电镀的均匀的细胞悬浮液，它包含大约70％的支持细胞也使用机械力（通过注射器针头或分别巴斯德吸管吸取消化管状片段反复）。后在培养3天后，使用介质是无血清的，支持细胞的百分比增加至大约90％。这可以在很大程度上归因于污染的生殖细胞的死亡。残余肾小管周围细胞（PTC）的，但是，坚定不移地通过自己的细胞外基质连接。的PTC，但不支持细胞，已知会产生纤连蛋白可以作为标志物蛋白用于与PTC的推定污染。因此，董建华等。理由是，一个附加的透明质酸酶治疗可以改善塞尔托利细胞级分7的纯度。事实上，他们可以表明，附加的处理是能够由PTC的，以减少污染约20倍，当在比较的含有血清的培养基中用于培养纯化塞尔托利细胞，其变得特别明显。从那时起Tung等的改进的过程。成为普遍的协议和外地8（图1B）被广泛地使用由其它主要基团。 在胶原酶处理多数的PTC的被释放并且可以并行地隔离，以支持细胞。而PTC的剧烈增殖，并且不向卵泡刺激素（FSH）反应，塞尔托利细胞不再进行有丝分裂和通过特性的形态变化到FSH响应和增加环磷酸腺苷（cAMP）的浓度9。非常相似的酶消化的协议可用于初级塞尔托利细胞从其他动物，如人10，鼠标11,12，西伯利亚仓鼠13或牦牛14的隔离。用于除去大量污染生殖细胞低渗休克可以在分离步骤15的端部可以采用。这也允许支持细胞的高效分离从成年大鼠睾丸16。一个富集塞尔托利细胞悬浮液，也可以从生殖细胞通过电镀涂有曼陀罗凝集素17凝集素菜悬浮液中分离。仅支持细胞和一些残留的PTC粘附到凝集素板。 原发性睾丸支持细胞培养，主要是从大鼠，已初步用于研究响应激素或建立细胞系，如鼠标支持细胞里NE TM4 18。该细胞系已在一百多个研究被调查直至今日。在平移的方法支持细胞已被用于共培养细胞和类似的在长期移植物存活异种或同种异体胰岛的共移植组织的免疫保护无全身免疫抑制19。分离的塞尔托利细胞已在共培养实验中被还用于研究上皮-间质（Sertoli细胞- PTCC）和体生殖细胞（支持细胞-生殖细胞）的相互作用20,21。最近主要支持细胞已被用于研究Toll样受体的表达和炎性细胞因子的分泌，以及信号转导级联，导致细胞因子感染后非致病性和肾盂肾炎大肠杆菌表达大肠杆菌 22。最近其他调查采用支持细胞为研究睾丸的免疫privilege 23和表明睾酮预处理抑制LPS诱导的炎症反应24。

<table border="0" cellpadding="0" cellspacing="0" width="813">
	<tbody>
		<tr height="80">
			<td height="80" style="height:80px;width:289px;">Actin (smooth muscle) antibody clone 1A4</td>
			<td style="width:111px;">Dako</td>
			<td style="width:124px;">M0851</td>
			<td style="width:289px;">Monoclonal mouse anti human antibody. 
			Use 1:100 dilution for immunofluorescence.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:289px;">Albumin bovine fraction V 
			Standard grade, lyophilized</td>
			<td style="width:111px;">Serva</td>
			<td style="width:124px;">11930.04</td>
			<td style="width:289px;">Filter (0.45 &#181;m) BSA solutions used for immunofluorescence.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">Corning&#8482; Cell Strainers 70&#160;&#181;m</td>
			<td style="width:111px;">Corning</td>
			<td style="width:124px;">431751</td>
			<td>White color</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:289px;">Collagenase A from Clostridium histolyticum</td>
			<td style="width:111px;">Roche</td>
			<td style="width:124px;">10103586001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">DAPI mountant ProLong&#174; Gold Antifade</td>
			<td style="width:111px;">Life technologies</td>
			<td style="width:124px;">P-36931</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">D-glucose</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:124px;">G8644</td>
			<td>100 g/l</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">Dulbecco&#180;s PBS without Ca2+/Mg2+</td>
			<td style="width:111px;">Gibco</td>
			<td style="width:124px;">14190-094</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">DNase I</td>
			<td style="width:111px;">Roche</td>
			<td style="width:124px;">10104159001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">Electron microscope</td>
			<td style="width:111px;">Zeiss</td>
			<td>EM 109S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">Fluorescence microscope</td>
			<td style="width:111px;">Zeiss</td>
			<td style="width:124px;">Axioplan 2</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">Hyaluronidase from bovine testis</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:124px;">H3506</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">Inverted light microscope</td>
			<td style="width:111px;">Olympus</td>
			<td style="width:124px;">CKX41</td>
			<td>Routine cell culture microscope</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:289px;">Mouse IgG / IgM (H+L) polyclonal 
			secondary Antibody&#160;</td>
			<td style="width:111px;">Life technologies</td>
			<td style="width:124px;">A-10684</td>
			<td>Alexa Fluor&#174; 488 conjugate</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:289px;">Oil Red O</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:124px;">O0625</td>
			<td style="width:289px;">Has replaced Sudan III and Sudan IV 
			because of brighter color.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">Paraformaldehyde</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:124px;">P6148</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:289px;">Penicillin (5,000&#160;U/ml)/ 
			Streptomycin (5,000 &#181;g/ml)</td>
			<td style="width:111px;">Gibco</td>
			<td style="width:124px;">15070-063</td>
			<td>100x solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">RPMI-1640</td>
			<td style="width:111px;">Gibco</td>
			<td style="width:124px;">21875-034</td>
			<td>Contains 300 mg/L L-glutamine</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">Trypsin from porcine pancreas</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:124px;">T5266</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">Trypan Blue Stain (0.4%)</td>
			<td style="width:111px;">Gibco</td>
			<td style="width:124px;">15250-061</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:289px;">Trypsin inhibitor from soybean</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:124px;">T6522</td>
			<td style="width:289px;">The Sigma product is considerably cheaper than the previously used 
			BPTI (Aprotinin) from Roche.</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:289px;">Vimentin antibody (V9)</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:124px;">V6630</td>
			<td style="width:289px;">Monoclonal mouse anti pig vimentin antibody.&#160; Use 1:100 dilution for immunofluorescence.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">Wistar WU rats</td>
			<td style="width:111px;">Charles River</td>
			<td style="width:124px;">N/A</td>
			<td>Should be 19 days old on day of experiment.</td>
		</tr>
	</tbody>
</table>

isolation of sertoli cells and peritubular cells from rat testes

睾丸，特别是雄配子，挑战以独特的方式免疫系统，因为精子的分化先出现在青春期的时候 - 建立全身免疫耐受十余年后。精细胞表达了许多可能被视为由免疫系统非自身蛋白质。然后在睾丸必须能够建立耐受这些新抗原一方面，但仍然能够保护自身免受感染和肿瘤的发展，另一方面。因此睾丸是体内该容忍外来抗原没有唤起一个有害的炎性免疫应答的少数免疫特权部位之一。支持细胞中发挥关键作用的维持睾丸的这种免疫特权环境，并且还延长cotransplanted细胞存活在外国的环境。因此主要支持细胞是研究睾丸无法通过电子邮件的免疫特权的重要工具通过建立的细胞系或其它细胞模型asily取代。在这里，我们提出了一个详细而全面的协议，用于支持细胞的分离 - 如果需要的管周细胞 - 从大鼠睾丸一天之内。

所描述的过程允许从10大鼠睾丸约12×10 7个塞尔托利细胞的分离。 3×10 6个细胞每孔接种于6孔板，使六至七个6孔板可用于在第7天实验胰DNA酶I消化是在塞尔托利细胞分离中最关键的步骤。如果在这点上消化正在推进太远，生殖细胞的比率/ Sertoli细胞会不成比例增加至结束。在培养3-6天它小心洗掉尽可能多的非粘性或松散连接的生殖细胞尽可能是很重要的。?...

Watch this Scientific Journal Video about Isolation of Sertoli Cells and Peritubular Cells from Rat Testes at JoVE.com

Isolation of Sertoli Cells and Peritubular Cells from Rat Testes

需要进行其免疫特性的炎症或感染，和利用在学习睾丸免疫特权，信号转导原塞尔托利细胞。在这里，我们描述了高度纯化的主支持细胞和从大鼠睾丸管周细胞的分离的基于酶的协议。

从大鼠睾丸支持细胞的分离和管周细胞

The testis, and in particular the male gamete, challenges the immune system in a unique way because differentiated sperm first appear at the time of ...

isolation-of-sertoli-cells-and-peritubular-cells-from-rat-testes

Research

JoVE Journal

Immunology and Infection

22.3K 次观看.  Justus-Liebig-Universität Gießen. 需要进行其免疫特性的炎症或感染，和利用在学习睾丸免疫特权，信号转导原塞尔托利细胞。在这里，我们描述了高度纯化的主支持细胞和从大鼠睾丸管周细胞的分离的基于酶的协议。 

视频: Isolation of Sertoli Cells and Peritubular Cells from Rat Testes

Isolation of Mouse Peritoneal Cavity Cells

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal tract and other viscera. It harbors a number of immune cells including macrophages, B cells and T cells. The presence of a high number of na&iuml;ve macrophages in the peritoneal cavity makes it a preferred site for the collection of na&iuml;ve tissue resident macrophages (1). The peritoneal cavity is also important to the study of B cells because of the presence of a unique peritoneal cavity-resident B cell subset known as B1 cells in addition to conventional B2 cells. B1 cells are subdivided into B1a and B1b cells, which can be distinguished by the surface expression of CD11b and CD5. B1 cells are an important source of natural IgM providing early protection from a variety of pathogens (2-4). These cells are autoreactive in nature (5), but how they are controlled to prevent autoimmunity is still not understood completely. On the contrary, CD5+ B1a cells possess some regulatory properties by virtue of their IL-10 producing capacity (6). Therefore, peritoneal cavity B1 cells are an interesting cell population to study because of their diverse function and many unaddressed questions associated with their development and regulation. The isolation of peritoneal cavity resident immune cells is tricky because of the lack of a defined structure inside the peritoneal cavity. Our protocol will describe a procedure for obtaining viable immune cells from the peritoneal cavity of mice, which then can be used for phenotypic analysis by flow cytometry and for different biochemical and immunological assays.

The peritoneal cavity in mammals contains different immune cell populations crucial for innate immune responses. An efficient isolation method is required for biochemical and functional analyses of these cells. Here we provide a comprehensive method for the isolation of peritoneal cavity cells in the mouse.

小鼠腹膜腔细胞的分离

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal ...

科研

免疫与感染

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 ...

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

在细胞命运<em&gt; E.大肠杆菌</em感染后的噬菌体λ

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Isolation of Mouse Lung Dendritic Cells

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

小鼠肺树突状细胞的分离

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and food antigens while concomitantly remaining poised to mount inflammatory responses toward invasive pathogens1,2. Antigen presenting cells, particularly DCs and macrophages, play critical roles in maintaining intestinal immune homeostasis via their ability to sense and appropriately respond to the microbiota3-14. Efficient isolation of intestinal DCs and macrophages is a critical step in characterizing the phenotype and function of these cells. While many effective methods of isolating intestinal immune cells, including DCs and macrophages, have been described6,10,15-24, many rely upon long digestions times that may negatively influence cell surface antigen expression, cell viability, and/or cell yield. Here, we detail a methodology for the rapid isolation of large numbers of viable, intestinal DCs and macrophages. Phenotypic characterization of intestinal DCs and macrophages is carried out by directly staining isolated intestinal cells with specific fluorescence-labeled monoclonal antibodies for multi-color flow cytometric analysis. Furthermore, highly pure DC and macrophage populations are isolated for functional studies utilizing CD11c and CD11b magnetic-activated cell sorting beads followed by cell sorting.

Here, we detail a methodology for the rapid isolation of mouse intestinal dendritic cells (DCs) and macrophages. Phenotypic characterization of intestinal DCs and macrophages is performed using multi-color flow cytometric analysis while magnetic bead enrichment followed by cell sorting is used to yield highly pure populations for functional studies.

从老鼠肠道树突状细胞和巨噬细胞的分离和表征

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

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 Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

从人类胸腺髓样树突状细胞和上皮细胞的分离

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

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 ...

Isolation of Murine Lymph Node Stromal Cells

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

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

鼠淋巴结基质细胞的分离

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

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

分离和流式细胞仪分析从缺血小鼠脑免疫细胞

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...