作者希望在森塔拉减肥中心的临床协调员贝基. 马奎斯的帮助下, 她的病人筛查和同意的过程。这项研究得到了 R15HL114062 对 Anca d Dobrian 的支持。

东弗吉尼亚医学院的机构审查委员会批准了研究和收集人体脂肪组织样本用于研究。从病人那里收集了知情的书面同意书。
1. 编制缓冲器、媒体和仪器
<ol><li>准备一个克雷布斯的碳酸氢钠-缓冲溶液 (KRBBS): 135 毫米氯化钠, 5 毫米氯化钾, 1 毫米硫酸镁, 0.4 毫米磷酸钾二元, 5.5 毫米葡萄糖, 1 毫米腺苷, 0.01% 抗生素/防霉混合 (50 µg/毫升青霉素,50µg/毫升链霉素, 30 µg/毫升庆大霉素, 15 ng/毫升两性霉素) 和10毫米 HEPES (pH = 7.4)。这个解决方案是准备新鲜的每一次的组织收集和消化。</li>
	<li>制备新的胶原酶溶液: 1 毫克/毫升的胶原酶, 类型 1, 在 KRBSS;做3毫升/克的脂肪。在组织消化前, 在水浴中预热37摄氏度的胶原酶溶液。</li>
	<li>准备一个 CD34 细胞隔离缓冲: 2% 胎牛血清 (血清) 和1毫米 EDTA 在无菌磷酸盐缓冲生理盐水 (PBS)。</li>
	<li>准备成脂培养基: DMEM/F12, 5% 血清, 50 µg/毫升青霉素/链霉素, 1.25 µL/毫升的人胰岛素 (5 µg/毫升或144亩/毫升), 0.36 µL/毫升2.8 毫米罗格列酮 (1 µM)。</li>
	<li>用0.3% 油红 O (破产) 库存溶液 (重量/容积) 将0.3 克的破产, 溶解为100毫升异丙醇。</li>
</ol>2. 脂肪间质血管分数隔离
注: 该研究包括一组2例病态肥胖型糖尿病 (T2D) 和非糖尿病患者, 年龄18–65岁, 在森塔拉代谢和减肥手术中心 (森塔拉医学小组, 诺福克, VA) 进行减肥手术。排除标准包括一种自身免疫性疾病, 包括1型糖尿病、需要长期免疫抑制剂治疗的条件、抗炎药物、thiazolinendiones、有效烟草使用、慢性或急性感染或历史在过去12月内治疗的恶性肿瘤。T2D 被定义为空腹血糖为126毫克/dl 或更大, 葡萄糖200毫克/dl 或更大后2小时葡萄糖耐受性测试, 或使用抗糖尿病药物。
<ol><li>收集人网膜 (OM) 和皮下 (SC) 脂肪组织 (AT) 从人体进行减肥手术。</li>
	<li>保持 OM 和 SC 在单独的小瓶包含汉克的缓冲盐溶液与50µg/毫升青霉素/链霉素在室温下立即组织提取。</li>
	<li>当样品在组织提取后到达实验室时, 用70% 乙醇擦拭剪刀和镊子仔细清洁和去除组织中的纤维化和烧灼部分。</li>
	<li>对脂肪组织进行称量, 将其分成5克 (或更少) 整除数胶原酶消化。</li>
	<li>用两对剪刀, 在室温闪烁的小瓶中, 在5毫升的胶原酶溶液中, 切碎5克。</li>
	<li>添加额外的10毫升胶原酶溶液的切碎组织为15毫升总。</li>
	<li>在连续晃动的水浴中, 将样品消化1小时, 摄氏37摄氏度。</li>
	<li>切断20毫升注射器的尖端, 使钝端。</li>
	<li>从250µm 网格中剪下一个9厘米 x 9 厘米的正方形, 用钝端注射器将其推到50毫升圆锥管中。</li>
	<li>将样品倒入20毫升钝端注射器中, 通过250µm 尼龙网过滤成50毫升锥形管, 将脂肪细胞和基质血管干细胞从未消化的组织中分离出来。 注: 不要使用超过5克的每50毫升圆锥管, 因为网将被堵塞由于过度纤维化未消化的材料。</li>
	<li>用10毫升的 KRBBS 冲洗出闪烁的瓶子, 然后通过过滤器将其倒入收集残余细胞。</li>
	<li>在室温下孵化过滤样品5分钟。 注意: 这一步是很重要的, 让脂肪细胞漂浮在管的顶部和一些基质血管干细胞, 以解决在管的底部。</li>
	<li>利用20毫升注射器和一个20克 x 6 在吹打针去除基质血管分数层, 并将其转移到一个干净的50毫升圆锥管。</li>
	<li>添加10毫升的 KRBBS, 并允许样品坐在板凳上5分钟, 在室温下。</li>
	<li>重复步骤2.12–2.14 两次。 注: 这些步骤将确保无污染的基质血管细胞与漂浮脂肪。</li>
	<li>将两个仓库的基质血管分数保持在冰上进行进一步处理, 如下面的步骤所述。 注意: 不要把细胞放在冰上超过1小时, 因为这可能会影响细胞的生存能力。脂肪细胞可以在液态氮中被闪光冷冻, 用于长期贮存或用于实验。</li>
</ol>3. 脂肪组织内皮细胞的分离
<ol><li>将基质血管馏分 (SVF) 在 500 x g处旋转5分钟, 在4摄氏度。</li>
	<li>从离心机上取出样品, 仔细地倒上清液;保持含有 SVF 细胞的颗粒。</li>
	<li>在5毫升的 PBS 中轻轻并用重悬细胞颗粒。 注: 如果5克以上的 a 在车厂被处理成多个整除数 (见步骤 2.4), 把细胞颗粒聚集在一起。</li>
	<li>旋转样品在 500 x g 5 分钟, 在4摄氏度。</li>
	<li>移除上清, 并用重悬1毫升的 PBS 细胞, 并计数细胞。 注: 在这里, 一个自动细胞计数器用于细胞计数。细胞的生存能力是通过混合12µL 细胞悬浮与12µL 的吖啶橙/碘碘 (AO/PI) 解决方案 (见材料表)。每个车厂每克的平均单元数为: (a) OM 车厂: 1.69 x 105 @ 4.51 x 104;(b) SC 车厂: 1.24 x 105 @ 6.45 x 104。两个仓库的细胞的生存能力是 > 90%。</li>
	<li>将样品按 500 x g分5分钟, 在4摄氏度。 注: CD34 磁选择协议 (见材料表) 已适应步骤3.7–3.15。</li>
	<li>并用重悬100µL CD34 隔离缓冲器中的颗粒。 注: 总细胞计数要求以下再悬浮容量: (a) < 2 x 107个细胞: 并用重悬颗粒在100µL;(b) 2 x 108–5 x 108细胞: 并用重悬在1毫升的颗粒。在步骤3.9–3.16 中, 所用试剂的体积缩小为 < 2 x 107细胞。</li>
	<li>添加10µL CD34 鸡尾酒, 并在室温下孵化样品15分钟。</li>
	<li>在室温下添加5µL 的磁性珠子, 并孵育样品10分钟。</li>
	<li>在每个样品中加入2.5 毫升的 CD34 隔离缓冲器, 将样品放入磁铁中, 在室温下放置5分钟。</li>
	<li>将磁铁反转5秒, 将隔离缓冲器倒掉。</li>
	<li>从磁铁中取出样品, 重复步骤3.10 和 3.11 4x。</li>
	<li>从磁铁中取出管子, 加入2毫升的 CD34 隔离缓冲器。</li>
	<li>将细胞以 500 x g为5分钟, 在4摄氏度。</li>
	<li>并用重悬细胞颗粒在1毫升的 CD34 隔离缓冲和计数细胞。 注: 在这里, 每克的平均细胞数是: (a) OM 车厂: 4.75 x 104 @ 3.36 x 104;(b) SC 车厂: 1.06 x 104 @ 9.53 x 103。CD31 塑料 immunobead 协议适用于步骤 3.16–3.34 (见材料表)。</li>
	<li>颗粒细胞在 500 x g 5 分钟和并用重悬的颗粒在250µL 的缓冲 B 和250µL 的洗涤缓冲器。</li>
	<li>涡流管彻底并用重悬 CD31 珠。</li>
	<li>添加20µL 的 CD31 珠。 注: 由于总单元数 > 1 x 106, 卷根据制造商的输入被缩小了。</li>
	<li>在室温下用摇摆30分钟孵化样品。</li>
	<li>将过滤器连接到无菌50毫升圆锥管。 注: 过滤器的较大开口必须在顶部。</li>
	<li>在细胞分离之前, 加入1毫升的洗涤缓冲器, 平衡过滤器。将步骤3.16 下生成的混合物应用于过滤器。</li>
	<li>用5毫升的洗涤缓冲器在圆形运动中清洗过滤器, 总容积为20毫升。将接头连接至无菌50毫升圆锥管, 并关闭鲁尔锁。</li>
	<li>将过滤器连接到接头。在滤网壁上添加1毫升洗涤缓冲器。在滤网壁上添加1毫升的活化缓冲 D。轻轻旋转样品, 在室温下孵化10分钟。</li>
	<li>添加1毫升的洗涤缓冲器。将细胞从珠子中分离出来, 吹打10x。 注意: 避免产生气泡。</li>
	<li>打开鲁尔锁, 允许分离的细胞流入50毫升锥形管。每次用1毫升的洗涤缓冲器清洗过滤器10x。</li>
	<li>丢弃接头和过滤器, 离心样品在 300 x g 10 分钟, 在4摄氏度。并用重悬细胞颗粒在2毫升完整的 EGM-2 培养基中进行培养。 注: 此时每克的平均细胞数为: (a) OM 车厂: 5.92 x 103 @ 4.27 x 103;(b) SC 车厂: 4.12 x 103 @ 2.1 x 103。两个仓库的细胞的生存能力超过90%。</li>
</ol>4. 成脂在离体内皮细胞中的诱导
<ol><li>在37摄氏度, 5% CO2孵化器中, 用完整的 EGM-2 培养基在6井组织培养处理的板材中生长细胞。每3–4天更换介质, 直到细胞达到80–90% 汇合。 注: 人口加倍是在2–3天之间。</li>
	<li>在100毫米培养皿上涂上细胞, 然后将细胞分成1:3 次, 然后将其展开。在这个阶段, 细胞在2通道上。</li>
	<li>使用自动单元格计数器计数单元格 (参见材料表)。</li>
	<li>种子大约100–200,000 细胞成6井板确保每个车厂重复井, 并培养他们在完整的 EGM-2 媒体2天。</li>
	<li>吸取任何生长培养基, 并用2毫升的 DMEM/F12 与5% 的血清和50µg/毫升的青霉素/链霉素的控制细胞, 并取代它与2毫升的成脂培养基 (见步骤 1.4) 的治疗细胞。</li>
	<li>在湿润 5% CO2孵化器中孵育37摄氏度的细胞13天, 每3到4天更换各自的培养基。 注: 在治疗4天后, 细胞将开始积聚脂质。</li>
	<li>根据公布的方法15-17进行破产和尼罗河红染色。</li>
	<li>要分离 RNA 提取的细胞, 从6井感应板中取出介质, 用1毫升的无菌 PBS 冲洗。</li>
	<li>添加350µL guanidium 硫氰酸盐-苯酚-氯仿溶液 (见材料表), 并在室温下孵化5–10分钟。</li>
	<li>用细胞刮刀刮掉盘子上的细胞, 将细胞转移到1.5 毫升的离心管上。 注: 样品可存储在-80 °c 冷冻库中, 用于 RNA 提取和以后进一步处理。</li>
	<li>使用一种适应的 RNA 萃取协议从样品中提取 mRNA (见材料表)。</li>
	<li>使用以下探针进行实时聚合酶链反应 (rt-pcr) 来评估基因表达: 脂联素、UCP-1 和 CIDEA (见材料表)。 注: 在这里, RT PCR 程序是采用以下标准协议: 变性 (95 °c; 十五年代), 退火和延长 (60 °c; 1 分钟) 40 循环。用 CT值 > 35 表达基因的样本被认为是无法检测的。</li>
</ol>

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作者没有什么可透露的。

本文的重点是为 CD34+CD31+ 内皮细胞从内脏和皮下的脂肪组织的分离、扩增和脂肪诱导提供一种方法。
已报告的方法, 以隔离从不同的啮齿动物或人类的血管床内皮细胞, 主要涉及使用 CD31 抗体荧光标记或耦合磁性珠18,19,20,21,22,23. 普遍使用这些隔离技术的一个主要挑战是不同血管层内皮细胞的异质性和成纤维细胞和血管壁细胞的高污染危险。然而, 对避免这种污染物的隔离技术进行了改进, 报告了24。利用这些技术, 大部分成熟的内皮细胞都是从组织中分离出来的。脂肪组织是一个巨大的茎/祖细胞库, 包括内皮祖25,26。一些论文报告了用 CD34+ 和 CD31+ 抗体11,27的组合纯化人体脂肪组织内皮细胞。表达两个标记的细胞是成熟内皮细胞和内皮祖11,28,29的混合种群。最近的一些开创性论文报告说, 脂肪血管内皮 (CD31+) 或壁画 (PDGFRβ +) 细胞的一个小子集是脂肪体细胞祖5,30的丰富来源。这些具有脂肪电位的血管细胞可以产生白色或褐色/米色脂肪, 并与脂肪血管5中的活性血管生成有关。这些发现可以提供新的治疗机会, 以改善肥胖的代谢表现和/或通过产生白色和/或热量脂肪细胞的血管祖细胞诱发体重减轻。因此, 有兴趣确定一种简便、经济的隔离、扩张和评估人体脂肪组织血管内皮细胞脂肪电位的方法。
人类皮下和网膜内脏脂肪组织库的 CD34+CD31+ 细胞, 以前的特点是根据其血管生成潜能, 炎症介质的产生, 其衰老标志物和其他功能11,31. 然而, 目前还没有关于这些细胞脂肪潜力的公开证据。以前的出版物, 使用磁性珠子分离 CD34+CD31+ 细胞报告从大量的主题 (10 或更多)11的细胞汇集的数据。本文首次报告了一种成功地从皮下和网膜库中分离和扩展来自单个人类捐献者的 CD34+CD31+ 细胞的协议。我们从脂肪组织的 2–3 g 中分离和扩张细胞。CD34+CD31+ 细胞代表3–5% 总 SVF 细胞和显示 > 90% 生存在隔离之后。这些细胞是高度增生, 并显示了广泛的反应, 对脂肪分化后, 与罗格列酮和胰岛素的体外刺激。以前的研究只使用脂肪干细胞或从人体脂肪组织的总基质血管分数来测试脂肪反应4,32,33,34。最近的一项研究使用了人体脂肪组织微血管的单细胞悬浮液, 但目前还不清楚分化脂肪细胞是否为3的壁画或内皮。
CD34+CD31+ 细胞的额外免疫分型使用流式细胞术显示他们缺乏 CD45 和 CD24 标记, 无论他们的来源地。重要的是, 我们表明, 在3通道的文化, CD31+CD34+ 细胞保留他们的内皮功能显示的乙酰 LDL 的积累和试管形成的3维矩阵。此外, 分化的脂肪细胞代表了白色和米色/褐色的混合种群, 根据其形态学和分子标记, 包括 UCP-1 蛋白表达。以前的研究表明, 脂肪组织血管可以是白色和棕色脂肪细胞的来源5,30和最近的数据显示了类似的结果, 关于分化的白色和功能热量米色细胞从人的脂肪血管3。
与大多数使用含有多种成分的脂肪鸡尾酒的出版物不同, 从体外祖细胞分化的脂肪细胞, 我们发现罗格列酮和胰岛素是必要的, 足够的脂肪CD34+CD31+ 细胞的诱导。虽然我们知道只有罗格列酮能诱发非脂肪细胞35的脂质积累, 但泛脂肪和褐色/米色脂肪的分子标记, 包括 UCP-1 蛋白在选择细胞中的表达, 证实了成熟一些分化细胞的脂肪表型。我们的双成分脂肪鸡尾酒简化了程序协议, 使其更具成本效益。
一个重要的观察是, CD34+CD31+ 细胞的脂肪反应是高度可变的捐献者和脂肪库。虽然在32、34之前报告了脂肪干细胞或基质血管祖细胞的脂肪反应中特定于车厂的差异, 但这种反应的主题变异性是一种新的观察。在分析的20例人体标本中, 3 SC 和 7 OM 细胞对脂肪诱导没有反应。最有力的反应显示 > 90% 的细胞响应诱导4样品从 SC 和2样品从 OM. 有趣的是, 我们发现反应与 HbA1c 呈负相关, 与对未分化细胞的体外胰岛素刺激。我们认为, 反应的差异并不是由于技术的重复性较差, 而是反映了模仿体内反应的细胞的个体指纹。保存这种差异反应需要进一步的验证, 可能是一种相对容易的筛选方法, 对治疗的反应, 旨在刺激成脂在体内。为了进一步了解脂肪电位中个体变异的来源, 需要额外的免疫分型来识别 CD34+CD31+ 种群中具有特定脂肪细胞祖功能的体细胞亚群。
本议定书的一些局限性包括细胞种群的不完全特征;较长的扩展和分化时间 (2 周 + 2 周);获取人体样本的潜在限制和需要立即处理新收集的组织;和目前缺乏功能性数据的分化细胞。这种方法有几个优点, 包括可重复的, 而不是劳动密集型的议定书;可能保留其体内表型的指纹的细胞;从极少量的组织中扩展细胞, 成本相对较低;在重新培养后 cryopreserve 细胞并保留其脂肪特征的可能性;还可以选择生成这些单元格的存储库, 以便用于将来的筛选或其他目的。
该协议和作为最终结果获得的 CD34+CD31+ 单元可以作为机械研究和筛选目的的平移平台使用。

最近的证据显示, 在小鼠和人类中, 在脂肪组织血管中的细胞子集可以被区分成白色或米色/褐色脂肪体1,2,3。这种细胞的表型是一个争议的主题, 有证据支持内皮细胞, 平滑肌/周细胞, 或一光谱的中间表型4,5,6,7。这种方法的发展范围是测试从肥胖人群中分离出不同脂肪库的 CD34+CD31+ 内皮细胞的脂肪电位。文献中的其他研究集中在脂肪电位的总基质血管分数或已知脂肪细胞祖细胞2,8,9。由于目前现有的技术可以专门针对脂肪组织内皮细胞的药物传递10, 了解这种细胞的潜力接受脂肪诱导向白色或米色脂肪体是重要的未来靶向治疗。
不同的组报告, CD31 和 CD34 标记作为代理人, 以隔离内皮细胞从人脂肪组织11,12,13。通常情况下, 隔离是使用两个连续步骤和一个使用磁性珠子的正选择来执行的。在本报告中, 利用 CD34+ 磁珠结合 CD31 塑料珠 immunoseparation。我们发现这种技术优于顺序磁性 immunoseparation 的保存典型的鹅卵石内皮形态学。此外, 我们能够产生足够的细胞, 以扩大和脂肪诱导开始从尽可能少的脂肪 g。小样本皮下脂肪活检足以为下游应用生产所需的细胞数量。这方面是潜在的重要, 特别是如果这一方法将被用于筛选对脂肪诱导的人的对象的反应。
与文献报道的其他系统不同, 这种方法仅利用两种成分脂肪诱导 CD34+CD31+ 细胞: PPARγ激动剂-罗格列酮-和人胰岛素。重要的是, 使用胰岛素的数量在正常/高范围内循环后吸收胰岛素的人14。Akt 磷酸化测定的细胞体外对胰岛素的反应程度与它们对诱导性鸡尾酒反应的能力没有关联。有趣的是, 使用这种诱导鸡尾酒和实验条件, 由白色和米色/褐色细胞的混合, 由细胞内脂质滴的大小和数量和分子标记的表达决定。这种简单和经济高效的诱导协议以及对应答器细胞表型的定量评估 (白色与米色) 允许筛选可能改变差异性米色平衡的药剂。: 白色脂肪细胞。
该方法还为了解人体脂肪组织中血管内皮祖细胞成脂的基本机制提供了一种平移方法。使用这种特殊的隔离/分化技术, 调查人员可以审问各种途径, 负责成脂在血管内皮细胞的子集, 从不同脂肪库的瘦身和肥胖的人。

isolation, expansion, and adipogenic induction of cd34+cd31+ endothelial cells from human omental and subcutaneous adipose tissue

肥胖伴随着脂肪组织的广泛重塑, 主要是通过脂肪细胞肥大。极端脂肪细胞的生长导致胰岛素、局部缺氧和炎症反应不良。通过刺激祖细胞功能性白细胞的分化, 可以预防脂肪细胞的自由基肥大, 从而可以改善脂肪组织的代谢健康, 同时减少炎症。另外, 通过刺激米色/褐色脂肪细胞的分化, 总能量消耗可以增加, 导致体重减轻。这种方法可以防止肥胖症的发病率, 如2型糖尿病和心血管疾病的发展。
本文描述了 CD31 和 CD34 标记的人脂肪组织内皮细胞的一个子集对白色和米色脂肪体的分离、扩增和分化。该方法相对便宜, 不劳动密集型。它需要进入人体脂肪组织和皮下仓库是适合取样。对于这个协议, 肥胖症患者的新鲜脂肪组织样本 [身体质量指数 (BMI) > 35] 是在减肥手术过程中收集的。使用序贯 immunoseparation 从基质血管的分数, 足够的细胞产生的小到 2–3 g 的脂肪。这些细胞可以在10–14天内进行培养, 可以冷冻保存, 并保留其脂肪的性质, 传代直至通过5–6。细胞被治疗14天与脂肪鸡尾酒使用人胰岛素和 PPARγ激动剂-罗格列酮的组合。
这种方法可用于获取分子机制的概念实验的证明, 推动脂肪内皮细胞的脂肪反应, 或筛选新的药物, 可以提高脂肪反应, 无论是对白色或米色/棕色脂肪细胞分化。使用小皮下活检, 这种方法可用于筛选非应答者的临床试验目的是刺激米色/棕色和白色脂肪细胞的治疗肥胖和共同疾病。

我们的协议旨在提供一种体外方法, 以确定 CD34+CD31+ 血管细胞从不同的储存库的人脂肪组织的脂肪电位。图 1A显示了简化流程图。第一步使用阳性选择的 CD34 表达细胞导致 > 95% CD34+ 细胞在人口的新鲜隔离细胞 (图 1A)。重要的是, 这个标记是丢失后, 细胞培养了几个段落。由于 CD34 是多种造血和非造血祖细胞的常见标记, 因此, 分离内皮祖的以下步骤是从 CD34+ 细胞人群中 CD31+ 的阳性选择 (图 1A)。虽然 CD31 是内皮细胞的标志物, 但也可以在造血细胞的亚群上找到。我们通过流式细胞术分析了网膜和皮下脂肪的几种制剂, 并一致发现 CD34+CD31+ 细胞不表达 CD45 标记 (图 1B, 顶部板)。通常情况下, < 1% 的细胞显示 CD45 阳性, 从总的细胞数量。这一结果使我们得出结论, 我们可能获得了几乎没有造血祖细胞的准备。为了确定细胞是否表达脂肪干细胞标记 CD24, 我们分析了网膜和皮下仓库的细胞, 发现几乎没有细胞表达 CD24 在网膜库中的标记 (图 1B, 底部板) 和少于5% 的细胞在皮下仓库中表达了标记 (未显示)。最后, 利用这种分离方法, 我们从网膜和皮下库的肥胖者中获得 CD34+CD31+CD45-CD24 细胞。
利用与 CD31 抗体共轭的塑料珠子, 我们获得了在早期通道显示鹅卵石内皮形态学的细胞, 而不是使用与 CD31 抗体共轭的磁性珠子分离的细胞, 显示出分离后立即形成纺锤形间质表型 (图 2A)。为了进一步证实 CD34+CD31+ 细胞的内皮特性, 我们进行了两种功能性的检测: DiI-低密度脂蛋白和基底膜基质 (简称基质) 试管形成的体外吸收。CD34+CD31+ 细胞以 DiI-低密度脂蛋白孵化, 绝大多数细胞为阳性 (图 1C)。作为一个积极的控制, 我们使用了人体脂肪组织微血管内皮细胞的主要细胞线 (HAMVEC), 这是商业可用 (见材料表和图 1C)。我们还测试了 CD34+CD31+ 细胞在培养后是否保留其内皮功能, 确定这些细胞在体外形成自发血管样结构的能力, 在3维矩阵中。在3通道后, CD31+CD34+ 形成类似于 HAMVEC 原发性内皮细胞线的基质中的管状容器结构 (图 1D)。
在2–3通道的细胞扩张后, 我们将细胞从 EGM-2 完整的培养基中转换为 DMEM/F12 培养基, 并辅以5% 的血清、罗格列酮 (1µM) 和胰岛素 (144 亩/毫升)。保持细胞在 EGM-2 完整的媒体大大减少了他们的脂肪分化。此外, 添加地塞米松和 3-isobuthyl-1 甲基黄嘌呤对培养基没有改变的速度和程度的脂积累和消炎痛的添加明显减少脂积累 (数据没有显示)。在这些初步实验的基础上, 我们决定只用罗格列酮和胰岛素进行脂肪诱导。经过14天的脂肪培养基, 细胞被固定和染色的油红色 O 或尼罗河红色和核复染与 DAPI (图 2B)。在这些数字中, 有代表性的图像显示的细胞从配对皮下 (SC) (图 2B, 顶部板) 和网膜 (图 2B, 底部板) 脂肪组织三人的体重指数之间的37–45公斤/米2。请注意, 对归纳的反应在主题和同一主题的仓库之间有很大的不同。如图 2B中的荧光图像所示, 单房性 (红色箭头) 和 multilocular (白色箭头) 的混合种群 (白箭) 含脂细胞以及不积累脂质的细胞 (黄色箭头) 通常都存在。这些细胞的数量之间的比例也高度可变的主题和来源库。对含脂细胞 (基于油红色 O 阳性率) 的百分比进行量化 (基于 DAPI 染色的细胞核) 表明同一个人的 sc 和 OM 库之间存在显著差异, 而 sc 库的细胞对脂肪感应更敏感 (图 2C)。对同一主体的主体与库间反应的异质性的解释还不清楚。然而, 我们推测, 这可能与携带体内表型/环境的指纹的细胞种群的固有性质有关, 而不是由于隔离协议的变异性。
为了证实细胞接受了分化为成熟的脂肪, 我们测量了泛脂肪标记脂联素的基因表达和棕色/米色标志物 UCP-1 和 CIDEA 在细胞后13天的脂肪诱导相比,非诱导的控制细胞。与对照组相比, 脂联素在分化细胞中的表达增加了1万倍 (图 2D)。CIDEA 和 UCP1 的基因表达只能在脂肪刺激后的细胞中检测到 (图 2D)。特别是, UCP-1 的表达是高度可变的, 反映了白色的不同比例: 棕色/米色细胞在细胞的数量。RPL27 基因的表达在第18-20 周期之间的 CT值之间表现出显著的一致性, 在脂肪分化的细胞和未经处理的控制。我们还检测到显示多室表型的细胞中的 UCP-1 蛋白表达, punctated 模式表明线粒体定位 (图 2E)。UCP-1 蛋白在没有积聚脂质的细胞中无法检测到, 或者细胞中有多个, 非常小的水滴, 很可能不是完全分化的脂肪组织 (图 2E)。
我们的观察, CD34+CD31+ 细胞的脂肪潜能是车厂特定和高度变量之间的不同学科, 促使我们寻求潜在的相关性 BMI, 年龄和 HbA1c。在测试的28例中, 我们没有发现脂肪电位与年龄或 BMI 之间有任何显著的相关性;然而, 我们确实发现了 HbA1c 与 OM 库细胞脂质积累的显著负相关 (图 3A)。这一发现表明与慢性高血糖环境有潜在的联系, 值得今后进行调查。这也表明, CD34+CD31+ 细胞可能携带在体内的签名, 他们的能力反应脂肪诱导。我们还表明, 这些细胞显示的变化水平的 Akt 磷酸化后,体外胰岛素刺激 (图 3B, 顶部)。然而, 这种反应的可变性与它们接受脂肪分化的能力并没有很好的关联, 如在匹配样品的图片中所示的油红色 O 染色 (图 3B, 底部)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57804/57804fig1.jpg"> 图 1: 人体脂肪组织中 CD34+CD31+ 细胞的分离和鉴定.(A) 流程图中显示了隔离和分化的主要步骤。在使用 CD34 磁珠进行阳性选择后, 在第二个选择步骤之前, > 95% 的新鲜隔离细胞 CD34+。(B)这种典型的流式细胞仪显示了活细胞种群的正向散射图封闭;FITC (BluFl2) 通道上的无瑕样本;和 CD45 染色的有代表性的网膜标本在上面。底部显示相同的序列, 显示在顶部, 但对 CD24 (Alexafluor 647 标记) 细胞从网膜样本。(C) 这些具有代表性的显微照片显示, CD34+CD31+ 细胞在4小时体外培养 (顶层) 中摄取 DiI-Ac 低密度脂蛋白 (红色)。在人体脂肪组织微血管内皮细胞原细胞系 (HAMVEC) (下板) 中发现相似的吸收。原子核由 DAPI 显示为蓝色。(D) 这些具有代表性的显微照片显示, 在3维矩阵中 CD34+CD31+ 细胞的试管形成。CD34+CD31+ 细胞 (通道 3) 在24井板上播种, FITC calcein 染色后, 在完全内皮细胞培养基中孵化4小时。这些细胞是用倒置荧光显微镜成像的。HAMVEC, 以相同的密度播种, 用于比较。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57804/57804fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57804/57804fig2.jpg"> 图 2: 脂肪诱导和 CD34+ CD31+ 细胞的标记.(A) 这些代表性显微照片显示了 CD34+CD31+ 细胞之间的形态学差异, 使用 CD31+ 磁性珠子与 CD31+ 塑料珠子的阳性选择相分离。细胞在隔离后的1通道被成像。使用的放大倍数为100X。(B) 这些小组是有代表性的显微照片的油红色 O 染色 CD34+CD31+ 细胞从配对皮下 (SC) 和网膜内脏 (OM) 样本3不同的对象。细胞培养为2–3通道在 EGM-2 完整的媒体, 然后打开 DMEM/F12 媒体与5% 血清和治疗胰岛素 (144 亩/毫升) 和罗格列酮 (1 µM) 14 天, 媒体改变每3天。使用的放大倍数为100X。代表荧光图像显示 adipo 红脂滴 (绿色) 和 DAPI 核染色 (蓝色)。请注意含有单房性脂 (红色箭头), multilocular 脂滴 (白色箭头) 的细胞组合, 或显示没有脂质积累的细胞 (黄色箭头)。所用的放大倍数为 200X, 刻度条 = 50 µm. (C) 本小组显示了脂肪电位的量化, 表示为油红 O (破产) 阳性细胞正常化为总 DAPI 染色核。同一学科的 OM 和 SC 库的 CD34+CD31+ 细胞显示, 配对学生的 t 检验 (n = 7) 对脂肪电位有显著的差异。(D) 在脂肪诱导和控制细胞后14天的细胞内, 用 rt-pcr 方法测定成熟脂肪酶 (脂联素、UCP-1 和 CIDEA) 的基因表达。这些数据被表达为脂联素基因表达的折变。CIDEA 和 UCP-1 的表达在对照样品中无法检测到。这些值代表ΔCt 规范化为 RPL27 作为管家基因。RLP27 的 CT值在第18-20 循环中包含在分析中的所有样本 (n = 3–5主题) 之间。数据以平均值表示。一个配对的学生的 t 检验被用来统计分析数据。p < 0.05 拒绝零假设。(E) 本小组显示了胰岛素和罗格列酮诱导13天后 CD34+CD31+ 细胞中 UCP-1 的蛋白表达。免疫细胞化学使用人多克隆 UCP-1 抗体显示了 UCP-1 的选择性表达在多个室脂肪。用罗丹明共轭二次抗体 (红色) 和 DAPI 染色对细胞核 (蓝色) 进行检测。在嵌入的大放大显示 punctated 红色信号对应于 UCP-1 (红色箭头), 以及不同大小的脂滴 (LD) 周围的细胞核 (N)。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57804/57804fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57804/57804fig3.jpg"> 图 3:脂肪分化、HbA1c 与体外胰岛素信号的相关性.(A) 对 OM 和 SC 细胞分别使用长矛 (非参数) 和皮尔逊 (参数) 相关系数来确定 HbA1c 与脂质积累之间的相关性 (n = 20)。零假说被拒绝了为p < 0.05。(B) 在顶部, 西方印迹显示磷酸化 Akt (绿色) 和总 Akt (红色) 表达在 CD34+CD31+ 细胞体外刺激 5 nM 胰岛素30分钟之前, 脂肪诱导 (n = 8)。在底部, 脂肪诱导14天后, 同一细胞的油红色 O 染色显示。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57804/57804fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue at JoVE.com

Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue

脂肪组织血管祖细胞中白、米色脂肪体的分化具有改善肥胖代谢的潜力。我们描述了 CD34+CD31+ 内皮细胞与人的脂肪分离的协议, 并为随后的体外扩增和分化为白色和米色脂肪。讨论了几种下游应用。

人网膜和皮下脂肪组织 CD34+CD31+ 内皮细胞的分离、扩增和脂肪诱导

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor ...

isolation-expansion-adipogenic-induction-cd34-cd31-endothelial-cells

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

11.7K 次观看.  Eastern Virginia Medical School. 脂肪组织血管祖细胞中白、米色脂肪体的分化具有改善肥胖代谢的潜力。我们描述了 CD34+CD31+ 内皮细胞与人的脂肪分离的协议, 并为随后的体外扩增和分化为白色和米色脂肪。讨论了几种下游应用。

视频: Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

人外周血NK细胞的扩展，纯化和功能评估

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

科研

免疫与感染

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the disease, is characterised by an accumulation of Plasmodium falciparum infected red blood cells (iRBC) at pigmented trophozoite stage in the microvasculature of the brain2-4. This microvessel obstruction (sequestration) leads to acidosis, hypoxia and harmful inflammatory cytokines (reviewed in 5). Sequestration is also found in most microvascular tissues of the human body2, 3. The mechanism by which iRBC attach to the blood vessel walls is still poorly understood.
The immortalized Human Brain microvascular Endothelial Cell line (HBEC-5i) has been used as an in vitro model of the blood-brain barrier6. However, Plasmodium falciparum iRBC attach only poorly to HBEC-5i in vitro, unlike the dense sequestration that occurs in cerebral malaria cases. We therefore developed a panning assay to select (enrich) various P. falciparum strains for adhesion to HBEC-5i in order to obtain populations of high-binding parasites, more representative of what occurs in vivo.
A sample of a parasite culture (mixture of iRBC and uninfected RBC) at the pigmented trophozoite stage is washed and incubated on a layer of HBEC-5i grown on a Petri dish. After incubation, the dish is gently washed free from uRBC and unbound iRBC. Fresh uRBC are added to the few iRBC attached to HBEC-5i and incubated overnight. As schizont stage parasites burst, merozoites reinvade RBC and these ring stage parasites are harvested the following day. Parasites are cultured until enough material is obtained (typically 2 to 4 weeks) and a new round of selection can be performed. Depending on the P. falciparum strain, 4 to 7 rounds of selection are needed in order to get a population where most parasites bind to HBEC-5i. The binding phenotype is progressively lost after a few weeks, indicating a switch in variant surface antigen gene expression, thus regular selection on HBEC-5i is required to maintain the phenotype.
In summary, we developed a selection assay rendering P. falciparum parasites a more "cerebral malaria adhesive" phenotype. We were able to select 3 out of 4 P. falciparum strains on HBEC-5i. This assay has also successfully been used to select parasites for binding to human dermal and pulmonary endothelial cells. Importantly, this method can be used to select tissue-specific parasite populations in order to identify candidate parasite ligands for binding to brain endothelium. Moreover, this assay can be used to screen for putative anti-sequestration drugs7.

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

An in vitro model for cerebral malaria sequestration is described1. Plasmodium falciparum infected red blood cells are selected for binding to immortalized human brain microvascular endothelial cells. The selected parasites show a distinct phenotype. The selection process can be applied using various P. falciparum strains and endothelial cell lines.

选择恶性疟原虫</emCytoadhesion>寄生虫对人脑血管内皮细胞

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the ...

Expansion of Human Peripheral Blood  &gamma;&delta; T Cells using Zoledronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and anti-infective activity.1 The majority of &gamma;&delta; T cells in peripheral blood have the V&gamma;9V&delta;2 T cell receptor. These cells recognize antigen in a major histocompatibility complex-independent manner and develop strong cytolytic and Th1-like effector functions.1Therefore, &gamma;&delta; T cells are attractive candidate effector cells for cancer immunotherapy. V&gamma;9V&#x03B4;2 T cells respond to phosphoantigens such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which is synthesized in bacteria via isoprenoid biosynthesis;2 and isopentenyl pyrophosphate (IPP), which is produced in eukaryotic cells through the mevalonate pathway.3 In physiological condition, the generation of IPP in nontransformed cell is not sufficient for the activation of &gamma;&delta; T cells. Dysregulation of mevalonate pathway in tumor cells leads to accumulation of IPP and &gamma;&delta; T cells activation.3 Because aminobisphosphonates (such as pamidronate or zoledronate) inhibit farnesyl pyrophosphate synthase (FPPS), the enzyme acting downstream of IPP in the mevalonate pathway, intracellular levels of IPP and sensitibity to &gamma;&delta; T cells recognition can be therapeutically increased by aminobisphosphonates. IPP accumulation is less efficient in nontransfomred cells than tumor cells with a pharmacologically relevant concentration of aminobisphosphonates, that allow us immunotherapy for cancer by activating &gamma;&delta; T cells with aminobisphosphonates. 4 Interestingly, IPP accumulates in monocytes when PBMC are treated with aminobisphosphonates, because of efficient drug uptake by these cells. 5 Monocytes that accumulate IPP become antigen-presenting cells and stimulate V&gamma;9V&delta;2 T cells in the peripheral blood.6 Based on these mechanisms, we developed a technique for large-scale expansion of &gamma;&delta; T cell cultures using zoledronate and interleukin-2 (IL-2).7 Other methods for expansion of &gamma;&delta; T cells utilize the synthetic phosphoantigens bromohydrin pyrophosphate (BrHPP)8 or 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP).9 All of these methods allow ex vivo expansion, resulting in large numbers of &gamma;&delta; T cells for use in adoptive immunotherapy. However, only zoledronate is an FDA-approved commercially available reagent. Zoledronate-expanded &gamma;&delta; T cells display CD27-CD45RA- effector memory phenotype and thier function can be evaluated by IFN-&gamma; production assay. 7

Expansion of Human Peripheral Blood  &#947;&#948; T Cells using Zoledronate

A method to expand &gamma;&delta; T cells from peripheral blood mononuclear cells (PBMC) is described. PBMC-derived &gamma;&delta; T cells are stimulated and expanded using zoledronate and interleukin-2 (IL-2). Large scale expansion of &gamma;&delta; T cells can be applied to autologous cellular immunotherapy of cancer.

扩展人外周血γδT细胞使用Zoledronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and ...

Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, neutrophils are the first inflammatory cells to migrate to the site of injury. Recruitment of neutrophils to an injury site is a stepwise process that includes first, dilation of blood vessels to increase blood flow; second, microvascular structural changes and escape of plasma proteins from the bloodstream; third, rolling, adhesion and transmigration of the neutrophil across the endothelium; and fourth accumulation of neutrophils at the site of injury2,3. A wide array of in vivo and in vitro methods has evolved to enable the study of these processes4. This method focuses on neutrophil transmigration across human endothelial cells.
One popular method for examining the molecular processes involved in neutrophil transmigration utilizes human neutrophils interacting with primary human umbilical vein endothelial cells (HUVEC)5. Neutrophil isolation has been described visually elsewhere6; thus this article will show the method for isolation of HUVEC. Once isolated and grown to confluence, endothelial cells are activated resulting in the upregulation of adhesion and activation molecules. For example, activation of endothelial cells with cytokines like TNF-&alpha; results in increased E-selectin and IL-8 expression7. E-selectin mediates capture and rolling of neutrophils and IL-8 mediates activation and firm adhesion of neutrophils. After adhesion neutrophils transmigrate. Transmigration can occur paracellularly (through endothelial cell junctions) or transcellularly (through the endothelial cell itself). In most cases, these interactions occur under flow conditions found in the vasculature7,8.
The parallel plate flow chamber is a widely used system that mimics the hydrodynamic shear stresses found in vivo and enables the study of neutrophil recruitment under flow condition in vitro9,10. Several companies produce parallel plate flow chambers and each have advantages and disadvantages. If fluorescent imaging is needed, glass or an optically similar polymer needs to be used. Endothelial cells do not grow well on glass.
Here we present an easy and rapid method for phase-contrast, DIC and fluorescent imaging of neutrophil transmigration using a low volume ibidi channel slide made of a polymer that supports the rapid adhesion and growth of human endothelial cells and has optical qualities that are comparable to glass. In this method, endothelial cells were grown and stimulated in an ibidi &mu;slide. Neutrophils were introduced under flow conditions and transmigration was assessed. Fluorescent imaging of the junctions enabled real-time determination of the extent of paracellular versus transcellular transmigration.

This article first describes a procedure for isolating human endothelial cells from umbilical veins and then shows how to use these cells to examine neutrophil transmigration under flow conditions. By using a low-volume flow chamber made from a polymer with the optical characteristics of glass, live-cell fluorescent imaging of rare cell populations is also possible.

分离人脐静脉内皮细胞和水流条件下其使用中的中性粒细胞轮回研究

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, ...

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 Leukocytes from the Human Maternal-fetal Interface

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua parietalis). This interface is the anatomical site of contact between maternal and fetal tissues; therefore, it is an immunological site of action during pregnancy. Infiltrating leukocytes at the maternal-fetal interface play a central role in implantation, pregnancy maintenance, and timing of delivery. Therefore, phenotypic and functional characterizations of these leukocytes will provide insight into the mechanisms that lead to&#160;pregnancy disorders. Several protocols have been described in order to isolate infiltrating leukocytes from the decidua basalis and decidua parietalis; however, the lack of consistency in the reagents, enzymes, and times of incubation makes it difficult to compare these results. Described herein is&#160;a novel approach that combines the use of gentle mechanical and enzymatic dissociation techniques to preserve the viability and integrity of extracellular and intracellular markers in leukocytes isolated from the human tissues at the maternal-fetal interface. Aside from immunophenotyping, cell culture, and cell sorting, the future applications of this protocol are numerous and varied. Following this protocol, the isolated leukocytes can be used to determine DNA methylation, expression of target genes, in vitro leukocyte functionality (i.e., phagocytosis, cytotoxicity, T-cell proliferation, and plasticity, etc.), and the production of reactive oxygen species at the maternal-fetal interface. Additionally, using the described protocol, this laboratory has been able to describe new and rare leukocytes at the maternal-fetal interface.

Described herein is a protocol to isolate and further study the infiltrating leukocytes of the decidua basalis and decidua parietalis - the human maternal-fetal interface. This protocol maintains the integrity of cell surface markers and yields enough viable cells for downstream applications as proven by flow cytometry analysis.

从人类母胎界面分离白细胞

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua ...

Isolation of Viral Replication Compartment-enriched Sub-nuclear Fractions from Adenovirus-infected Normal Human Cells

During infection of human cells by adenovirus (Ad), the host cell nucleus is dramatically reorganized, leading to formation of nuclear microenvironments through the recruitment of viral and cellular proteins to sites occupied by the viral genome. These sites, called replication compartments (RC), can be considered viral-induced nuclear domains where the viral genome is localized and viral and cellular proteins that participate in replication, transcription and post-transcriptional processing are recruited. Moreover, cellular proteins involved in the antiviral response, such as tumor suppressor proteins, DNA damage response (DDR) components and innate immune response factors are also co-opted to RC. Although RC seem to play a crucial role to promote an efficient and productive replication cycle, a detailed analysis of their composition and associated activities has not been made. To facilitate the study of adenoviral RC and potentially those from other DNA viruses that replicate in the cell nucleus, we adapted a simple procedure based on velocity gradients to isolate Ad RC and established a cell-free system amenable to conduct morphological, functional and compositional studies of these virus-induced subnuclear structures, as well as to study their impact on host-cell interactions.

We provide a novel strategy to isolate viral replication compartments (RC) from adenovirus (Ad)-infected human cells. This approach represents a cell-free system that can help to elucidate the molecular mechanisms regulating viral genome replication and expression as well as regulation of viral-host interactions established at the RC.

由腺病毒感染正常细胞分离病毒复制车厢富集亚核分数

During infection of human cells by adenovirus (Ad), the host cell nucleus is dramatically reorganized, leading to formation of nuclear microenvironments ...

Isolation of Sertoli Cells and Peritubular Cells from Rat Testes

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 puberty - more than ten years after the establishment of systemic immune tolerance. Spermatogenic cells express a number of proteins that may be seen as non-self by the immune system. The testis must then be able to establish tolerance to these neo-antigens on the one hand but still be able to protect itself from infections and tumor development on the other hand. Therefore the testis is one of a few immune privileged sites in the body that tolerate foreign antigens without evoking a detrimental inflammatory immune response. Sertoli cells play a key role for the maintenance of this immune privileged environment of the testis and also prolong survival of cotransplanted cells in a foreign environment. Therefore primary Sertoli cells are an important tool for studying the immune privilege of the testis that cannot be easily replaced by established cell lines or other cellular models. Here we present a detailed and comprehensive protocol for the isolation of Sertoli cells - and peritubular cells if desired - from rat testes within a single day.

Primary Sertoli cells are required for studying testis-immune privilege, signal transduction during inflammation or infection, and utilization of their immunoprotective properties. Here we describe an enzyme-based protocol for the isolation of highly purified primary Sertoli cells and peritubular cells from rat testes.

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 Macrophage Subsets and Stromal Cells from Human and Mouse Myocardial Specimens

Macrophages represent the most heterogeneous and abundant immune cell populations in the heart and are central in driving inflammation and reparative responses after cardiac injury. How various subsets of macrophages orchestrate the immune responses after cardiac injury is an active area of research. Presented here is a simple protocol that our lab performs routinely, for the extraction of macrophages from mouse and human myocardium specimens obtained from healthy and diseased individuals. Briefly, this protocol involves enzymatic digestion of cardiac tissue to generate a single cell suspension, followed by antibody staining, and flow cytometry. This technique is suitable for functional assays performed on sorted cells as well as bulk and single cell RNA sequencing. A major advantage of this protocol is its simplicity, minimal day to day variation and wide applicability allowing investigation of macrophage heterogeneity across various mouse models and human disease entities.

Presented here is a protocol to isolate various subsets of macrophages and other non-immune cells from human and mouse myocardium by preparing a single cell suspension through enzymatic digestion. Gating schemes for flow cytometry based identification and characterization of isolated macrophages are also presented.

从人类和小鼠心肌标本中分离巨噬细胞子集和细胞位细胞

Macrophages represent the most heterogeneous and abundant immune cell populations in the heart and are central in driving inflammation and reparative ...