The authors would like to thank Joshua Kimani, clinical director of the research program at the University of Nairobi, for his assistance with mucosal immunology studies related to this protocol. The authors would like to acknowledge funding from CHVI grant MOP 86721.

伦理声明：曼尼托巴大学及内罗毕肯雅塔的国家医院/大学研究伦理委员会批准了这项研究，并签署知情同意从所有研究参与者获得。 媒体1。准备和CMC收集管<ol><li>制备磷酸盐缓冲盐水（PBS）溶液（137.93 mM氯化钠，2.67毫米氯化钾，8.1毫米的Na 2 HPO 4，1.47 mM的KH 2 PO 4）。高压灭菌无菌。这可以被存储在4℃几个月。 </li><li>预等分5毫升PBS入50ml Falcon管（每个样品1被收集），并在4℃储存直至样品收集时间。 </li><li>标签每个样品2-4冻存管或1.5 ml离心管中，以收集灌洗液进行存储。 </li><li>制备细胞培养基：RPMI 1640 +1％青霉素/链霉素/两性霉素B（终浓度为100单位/毫升，100微克/毫升和250纳克/毫升，respectiv伊利，没有FC / FB的补充。 </li></ol>细胞刷样品2。收藏收集细胞刷样本是必须由一个受过训练的医师或妇科医生进行非侵入性的过程。 <ol><li>同时使用细胞刷和一个木制或塑料刮板收集宫颈单个核细胞（CMC）。通过将刮刀与周围的宫颈内口的旋转（ 图1）收集的CMC从下窥器检查参与者。将细胞刷入颈管口，旋转360°，并立即放置两个细胞刷和刮具入含有5ml的PBS 50ml的falcon管。 </li><li>保持样品在冰/ 4℃，直到处理。为了保持细胞活力，处理样品在2小时内收集的。 </li><li>如果可能的话，收集绿色的顶部肝素真空采血管的外周血单个核细胞（PBMC）中分离，作为一个比较器/控制CMC分析匹配的血液样本。 </LI&gt; </ol>中医诊所3。隔离在生物安全二级实验室进行样品处理，在无菌生物安全柜，双层手套。 <ol><li>在收集，中央军委样本可能被污染的血液。排除具有从研究可见血液污染的样品，以防止混淆列入PBMC的最终样品中。由于淋巴细胞管委会样本中分离出的低号，次血液污染可以很容易地压倒管委会淋巴细胞组成部分。 </li><li>涡猎鹰管含有的细胞刷及刮刀45秒，从刷游离细胞。在此过程中样品可能成为泡沫状;这是正常的。 </li><li>使用细胞刷刮任何遗留物质掉刮板，并丢弃刮成漂白剂溶液。收集剩余附着在刷/刮刀的任何单元格，使用新鲜的手套挤拇指和食指之间的刮刀。滑下刷，使细胞和PBS，并收集在同一个50ml管中。丢弃细胞刷成漂白剂溶液，并丢弃的手套。 重要提示：使用一个新的手套对每个样品。 </li><li>适合100微米尼龙细胞过滤到新的50ml falcon管。使用移液管，收集从样品管中的PBS-细胞悬浮液，并通过过滤器到干净试管中进行过滤。 </li><li>加入5 ml的RPMI 1640中，以原始样品管中。使用移液管，洗样品管的侧面与RPMI，并通过过滤器转移到新的收集管。洗尼龙过滤器的RPMI的底部为好。 </li><li>离心样品10分钟，在514×g下。关键的是要在该步骤中离开离心机制动切断，以防止在CMC粒料撞出。 </li><li>轻轻地从管取出上清液，注意不要打扰颗粒。颗粒大小会变化很大;在这样我的样品，将沉淀是由于大量上皮细胞的存在相当大的，而在其他情况下，将丸粒是勉强可见和高透明度。 </li><li>由猎鹰管轻轻搅动悬浮颗粒。加入5毫升的PBS洗涤样品。 </li><li>再次离心在514 XG没有离心制动器10分钟。 </li><li>小心弃去上清液并重新悬浮沉淀如上述。这些细胞是准备或者冷冻保存（使用PBMC冷冻保存协议），刺激或流式细胞仪分型。 </li></ol> 4，CMC表面染色和流式细胞仪<ol><li>从在100μl封闭液的步骤3.10重悬沉淀并转移要么到96孔板或FACS管。封闭液（方框Fcγ受体）的配方如下：1.8微升小鼠IgG（最终浓度0.2微克/微升），5微升的FBS和93.2微升FACS洗涤液（PBS +2％FBS）中。染色可在96孔板中进行，如果粒料的大小是足够小的，但可能需要在FACS管中进行，如果颗粒是常规大。它相应滴定抗体是很重要的。 </li><li>阻断样品在冰上/ 10分钟，在4℃下</li><li>在96孔板中加入100μlFACS洗涤液（PBS +2％FBS）中，或加入500μl在FACS管洗​​涤细胞。离心机在600×g下在4℃下10分钟，使用于低制动（或关闭，如果低速制动器设置是不可用的）。 </li><li>除去上清液和重悬细胞沉淀通过搅拌。添加活力染色（现场死可以解决的可行性染料）。根据制造商的说明制备的可行性染料。它可能需要被滴定为在每个实验/流式细胞仪的设置使用。孵育在黑暗中30分钟，在4℃下</li><li>洗涤细胞100μl/500微升PBS（对于96孔板/ FACS管）。离心600×g离心10分钟，低/无刹车。删除supernatan吨，重悬细胞。 </li><li>孵育细胞表面标记的抗体的混合物，在100微升的总体积。重要提示：优化和滴定样品染色前各流式细胞仪面板。孵育细胞在黑暗中30分钟，在4℃下</li><li>重复步骤4.5。如果染色在96 - 孔板中，细胞转移到FACS管中，并稀释至750微升FACS洗涤的含1％多聚甲醛（PFA）的最终体积（或CytoFix，稀释1中4）。进行数据采集的流式细胞仪。 </li></ol> 5，收集和宫颈阴道灌洗的制备（CVL） <ol><li>此前细胞刷取样，用注射器2毫升无菌PBS冲洗宫颈内口和阴道后穹窿吸出灌洗。 </li><li>吸气式灌洗样品收集到15毫升猎鹰管并保持在冰上/ 4℃，直到处理。 </li><li>在400×g离心7分钟，以除去细胞碎片离心样品。 </li><li>合作llect上清液，等分试样的样品为1毫升或500微升的等分试样，并储存于-70℃。等分可以解冻ELISA或CVL蛋白微球阵列分析，但要避免多次冻结/解冻周期。 </li><li>如果需要的话，重悬细胞碎片的沉淀物中的RNA后来由以下制造商的方案来测量RNA表达。 </li></ol> 6，数据采集<ol><li>准备一套补偿管的抗体面板相匹配。运行补偿管调节光谱重叠。运行样品上的任何多色流式细胞仪被配置在面板中使用的荧光标记抗体。 </li><li>首先获取PBMC样本调整前向散射（FSC）和侧向散射光（SSC）电压，以检测淋巴细胞群。尽量让他们在100上每个轴的线性刻度。运行PBMC控制将使显著更容易找到管委会淋巴细胞群。 </li><li>对于CMC样品FSC的门槛可能需要相对增加至因涂抹了SSC轴在低FSC值PBMC样品。 </li><li>收购之前，每个涡管。获得整个管最大化收集淋巴细胞门事件的数目。监控机器小心，以免堵塞。 </li><li>将数据导出到流式细胞仪分析程序，如FlowJo。 </li></ol> 7，门控策略<ol><li>选择正向侧向散射高（FSC-H）/正向侧向散射面积（FSC-A）来确定单人口。 </li><li>选择时间与荧光标记（如例如FITC）来控制收购质量。变化的流量可能引入的数据失真。排除显示差异流速的任何区域。 </li><li>标识上SSC-A/FSC-A情节淋巴细胞群。用PBMC控制，以方便识别。需要注意的是管委会淋巴细胞群可能不一样清晰的PBMC控制。&lt;/ LI&gt; <li>门上SSC-A/viability染料排除死细胞与活细胞。死细胞可以非特异性结合的抗体，其中将介绍文物。 </li><li>门上的SSC-A/CD3识别的T细胞群体。从这个门，确定CD4 +和CD8 +的人群。 </li></ol>

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The authors have nothing to disclose.

面对庞大的知识差距的相对免疫力在女性生殖道（FGT），中医诊所的表型分析可以提供见解的各种各样成多个淋巴细胞群在子宫颈​​。再加上蛋白质组分析及在宫颈灌洗病毒载量的测量，抗干扰性性传播感染（STI）s和其他病原体可在解剖不同人群。 技术因素-中医诊所：隔离和CMC样品成功染色是具有挑战性的。在CMC收集协议的优化强调第一收集CVL的有效性，随...

大多数新感染艾滋病毒引起全世界通过异性性接触传染，女性占新发感染的47％，2011年（联合国艾滋病规划署1）。了解女性生殖道（FGT），主入口门户为HIV和其他性传染病原体之一，是的路径，寻找有效的策略，以防止感染的高度重视。在生殖器粘膜免疫应答是独特的清楚和从那些在外周血2测得不同。然而，在FGT免疫动力学的当前的知识是在最好的限定。迄今为止，黏膜免疫环境的研究主要集中在肠道相关淋巴组织（GALT），它已经很清楚，感染后粘膜组织中的早期事件，对后续的疾病进展3,4的强烈冲击。从生殖器粘膜采集样本代表一个巨大的挑战，并至少部分负责LACk的FGT的免疫学的理解。解决在不同的环境，是FGT就必须有效的方法从该地区采集和分析样品的情况下主机和病原体之间的免疫动态的难题。 在FGT被分成两部分：上部生殖道，包括输卵管，子宫内膜和宫颈管，下部尿路含有宫颈和阴道（由Kaushic评论等 5）。现在还不清楚是什么，这些不同的网站的相对贡献是艾滋病毒感染，但据信，这两个网站可能导致艾滋病毒进入6。 T细胞代表在上下生殖道的白细胞的40-50％，而巨噬细胞含有约10％（以罗德里格斯加西亚评论等 2）。 T细胞可以在阴道，子宫颈，子宫内膜和被检测到。巨噬细胞是更强烈的局部性ZED的子宫内膜和肌层结缔组织比子宫颈，虽然它们可以同时在组织中被检测到。最后，浆细胞样树突细胞（PDC）和朗格汉斯细胞也可以在FGT组织中检测到。表型和免疫人群及其易于感染艾滋病毒的比例可根据荷尔蒙周期变化重要的是，使用荷尔蒙避孕药，细菌性阴道炎，或性活动5,7-9的。 多种方法已经被开发，研究免疫人群的FGT和环境。宫颈活检，宫颈cytobrushes和宫颈阴道灌洗液（CVL）10-12是整个文献中最常用的。 CVL收集由PBS灌洗是最简单的方法，并允许免疫调节蛋白，但结果在极低的细胞产量的研究中，并因此不适合用于研究FGT 13的免疫细胞群。 CVL样品，另一方面，VERY用于通过测量用的方法，如ELISA，细胞因子微球阵列14或质谱15,16的各种细胞因子，趋化因子或抗微生物因子的表达评估FGT的免疫环境是有用的。免疫细胞频率，表型和功能特性可通过宫颈细胞刷或宫颈活检取样采集子宫颈单个核细胞（CMC）来实现。 宫颈活检取样是一种侵入性方法，其增加出血的不适和风险，需要2至11天治愈以下的步骤取决于女人12的免疫状态。另一方面，宫颈cytobrushes，尽管收集的细胞的产量较低，是一种微创的，更方便的方法来收集免疫细胞从FGT。这两种方法都可以达到的CD45 +白细胞中相同的产率，但是两个连续的子cytobrushes是必要的，得到的细胞相同数量的载一n一个活检13。然而，细胞刷取样仍然为进一步体外表型通过流式细胞仪14提供了细胞的可接受的数量（大约5,000的CD45 +细胞/细胞刷）。此外，功能特性，可以进行对这些样品，如刺激和细胞内流式细胞术定量PCR或已使用细胞刷衍生的CMC识别HIV特异性免疫应答17或Th细胞极化18执行。扩张性T细胞的人口也可方便与中医诊所19功能研究。 要注意的是活检和cytobrushes采样FGT不同的部分是很重要的。活检是从宫颈12,13的上皮和基质的优越部导出，而颈cytobrushes采样宫颈内口，收集从宫颈管和推测的转化区的上皮来源的细胞。因此，细胞刷样品品尝重新只园组成的柱状上皮单层的，而活检，包括由鳞状复层上皮衬里5的区域。其结果是，通过宫颈活检和细胞刷收集的白细胞种群的性质不同而不同。活检收集CD3 + T细胞的比例较高，而cytobrushes导致收集的CD14 +单核细胞/巨噬细胞13的比例较高。 研究FGT的免疫学一直是关注了很多年20-22，我们已经积累了大量的专业知识与细胞刷源性间中医诊所的研究。我们的研究重点主要在艾滋病病毒感染，感染和HIV阴性曝光（HESN）女性性工作者来自肯尼亚内罗毕的研究。优先病毒复制在活化的T细胞23和较低的数字活化细胞，可以由艾滋病病毒在FGT有针对性有助于防止感染HIV。根据这一假设，几个STUDI线ES形容HESN性工作者谁是高度暴露于艾滋病毒在较低的免疫激活但仍然未感染的24,25，和这个静态的表型是在FGT 14还观察到。在这里，我们描述的方法进行处理，并通过评估体外流式细胞仪来源于宫颈cytobrushes CMC样品中的T细胞活化。

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			<td height="21" style="height:21px;width:295px;">Name of Material/ Equipment</td>
			<td style="width:136px;">Company</td>
			<td style="width:133px;">Catalog Number</td>
			<td style="width:205px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">100uM Cell Strainer for 50 ml Falcon tube</td>
			<td style="width:136px;">BD</td>
			<td style="width:133px;">352360</td>
			<td>CMC processing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">RPMI 1640</td>
			<td style="width:136px;">Hyclone</td>
			<td style="width:133px;">SH30027.01</td>
			<td>CMC processing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Fetal Bovine serum&#160;</td>
			<td style="width:136px;">Life technology</td>
			<td style="width:133px;">16000044</td>
			<td>CMC processing</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Fungizone</td>
			<td style="width:136px;">Life technology</td>
			<td style="width:133px;">15290-018</td>
			<td>CMC processing</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Penicillin/streptomycin</td>
			<td style="width:136px;">Sigma</td>
			<td style="width:133px;">P4333-20ml</td>
			<td>CMC processing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">50ml Falcon tube</td>
			<td style="width:136px;">Fisher</td>
			<td style="width:133px;">14-959-49A</td>
			<td>CMC processing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Blood Bank disposable transfer pipette</td>
			<td style="width:136px;">Fisher&#160;</td>
			<td style="width:133px;">13-711-6M</td>
			<td>CMC processing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Cytobrush plus</td>
			<td style="width:136px;">Cooper surgical</td>
			<td style="width:133px;">C0121</td>
			<td>CMC sampling</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Disposable cervical scraper</td>
			<td style="width:136px;">Quick medical</td>
			<td style="width:133px;">2183</td>
			<td>CMC sampling</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">15 ml Falcon tube&#160;</td>
			<td style="width:136px;">Fisher&#160;</td>
			<td style="width:133px;">14-959-70c</td>
			<td>CVL processsing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">1.5ml tube ependroff</td>
			<td style="width:136px;">Fisher</td>
			<td style="width:133px;">05-402-18</td>
			<td>CVL storage</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LIVE/DEAD Fixable Cell Stain Kit</td>
			<td style="width:136px;">Invitrogen</td>
			<td>Various</td>
			<td>Flow cytometry reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fixation Buffer (4% PFA)</td>
			<td style="width:136px;">BD</td>
			<td>554655</td>
			<td>Flow cytometry regeant&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">IgG mouse&#160;</td>
			<td style="width:136px;">Sigma</td>
			<td style="width:133px;">I8765</td>
			<td>Flow cytometry regeant&#160;</td>
		</tr>
	</tbody>
</table>

collection, isolation, and flow cytometric analysis of human endocervical samples

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female genital tract (FGT). Because heterosexual transmission now represents the dominant mechanism of HIV transmission, and given the continual spread of sexually transmitted infections (STIs), it is critical to understand the interplay between host and pathogen at the genital mucosa. The substantial gaps in knowledge around FGT immunity are partially due to the difficulty in successfully collecting and processing mucosal samples. In order to facilitate studies with sufficient sample size, collection techniques must be minimally invasive and efficient. To this end, a protocol for the collection of cervical cytobrush samples and subsequent isolation of cervical mononuclear cells (CMC) has been optimized. Using ex vivo flow cytometry-based immunophenotyping, it is possible to accurately and reliably quantify CMC lymphocyte/monocyte population frequencies and phenotypes. This technique can be coupled with the collection of cervical-vaginal lavage (CVL), which contains soluble immune mediators including cytokines, chemokines and anti-proteases, all of which can be used to determine the anti- or pro-inflammatory environment in the vagina.

多参数流式细胞仪是一种功能强大的工具来剖析细胞亚群的表型和功能在以前未知的组织。 CMC样品的分析可以产生两个淋巴细胞和单核细胞的人口信息与相应的门控策略。 一位代表中央军委门控策略，相比匹配的PBMC轮廓， 如图2，FSC-A与FSC-H的情节使得细胞双峰，这是非常普遍的CMC样品相比，外周血单个核细胞的排斥，甚至是过滤后（ 图2A）。质量?...

Watch this Scientific Journal Video about Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples at JoVE.com

Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples

The use of cytobrush sampling to collect lymphocytes and monocytes from the endocervix is a minimally invasive technique that provides samples for analysis of female genital tract immunity. In this protocol, we describe the collection of cytobrush samples and immune cell isolation for flow cytometry assays.

收集，分离，和人类宫颈样品的流式细胞仪分析

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female ...

collection-isolation-flow-cytometric-analysis-human-endocervical

Research

JoVE Journal

Medicine

19.0K 次观看.  University of Manitoba. The use of cytobrush sampling to collect lymphocytes and monocytes from the endocervix is a minimally invasive technique that provides samples for analysis of female genital tract immunity. In this protocol, we describe the collection of cytobrush samples and immune cell isolation for flow cytometry assays. 

视频: Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples

Isolation of Human Islets from Partially Pancreatectomized Patients

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 diabetic islets from organ donors2. Here we share our protocol for isolating islets from human pancreatic tissue obtained from type 2 diabetic and non-diabetic patients who have undergone partial pancreatectomy due to different pancreatic diseases (benign or malignant pancreatic tumors, chronic pancreatitis, and common bile duct or duodenal tumors). All patients involved gave their consent to this study, which had also been approved by the local ethics committee. The surgical specimens were immediately delivered to the pathologist who selected soft and healthy appearing pancreatic tissue for islet isolation, retaining the damaged tissue for diagnostic purposes. We found that to isolate more than 1,000 islets, we had to begin with at least 2 g of pancreatic tissue. Also essential to our protocol was to visibly distend the tissue when injecting the enzyme-containing media and subsequently mince it to aid digestion by increasing the surface area.
To extend the applicability of our protocol to include the occasional case in which a large amount (&gt;15g) of human pancreatic tissue is available , we used a Ricordi chamber (50 ml) to digest the tissue. During digestion, we manually shook the Ricordi chamber3 at an intensity that varied by specimen according to its level of tissue fibrosis. A discontinous Ficoll gradient was then used to separate the islets from acinar tissue. We noted that the tissue pellet should be small enough to be homogenously resuspended in Ficoll medium with a density of 1.125 g/ml. After isolation, we cultured the islets under stress free conditions (no shaking or rotation) with 5% CO2 at 37 &deg;C for at least 48 h in order to facilitate their functional recovery. Widespread application of our protocol and its future improvement could enable the timely harvesting of large quantities of human islets from diabetic and clinically matched non-diabetic subjects, greatly advancing type 2 diabetes research.

The supply of type 2 diabetic islets for research is insufficient. Here we share our protocol for isolating islets from patients undergoing partial pancreatectomy. This approach represents a unique venue for obtaining islets from type 2 diabetic and clinically matched non-diabetic subjects in adequate numbers for basic and clinical studies.

从部分Pancreatectomized患者的人类胰岛的分离

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 ...

科研

医学

Collection Protocol for Human Pancreas

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation of pancreas recovered from cadaveric organ donors. The pancreas is divided into 3 main regions (head, body, tail) followed by serial transverse sections throughout the medial to lateral axis. Alternating sections are used for fixed paraffin and fresh frozen blocks and remnant samples are minced for snap frozen sample preparations, either with or without RNAse inhibitors, for DNA, RNA, or protein isolation. The overall goal of the pancreas dissection procedure is to sample the entire pancreas while maintaining anatomical orientation.
Endocrine cell heterogeneity in terms of islet composition, size, and numbers is reported for human islets compared to rodent islets 1. The majority of human islets from the pancreas head, body and tail regions are composed of insulin-containing &beta;-cells followed by lower proportions of glucagon-containing &alpha;-cells and somatostatin-containing &delta;-cells. Pancreatic polypeptide-containing PP cells and ghrelin-containing epsilon cells are also present but in small numbers. In contrast, the uncinate region contains islets that are primarily composed of pancreatic polypeptide-containing PP cells 2. These regional islet variations arise from developmental differences. The pancreas develops from the ventral and dorsal pancreatic buds in the foregut and after rotation of the stomach and duodenum, the ventral lobe moves and fuses with the dorsal 3. The ventral lobe forms the posterior portion of the head including the uncinate process while the dorsal lobe gives rise to the rest of the organ. Regional pancreatic variation is also reported with the tail region having higher islet density compared to other regions and the dorsal lobe-derived components undergoing selective atrophy in type 1 diabetes4,5.
Additional organs and tissues are often recovered from the organ donors and include pancreatic lymph nodes, spleen and non-pancreatic lymph nodes. These samples are recovered with similar formats as for the pancreas with the addition of isolation of cryopreserved cells. When the proximal duodenum is included with the pancreas, duodenal mucosa may be collected for paraffin and frozen blocks and minced snap frozen preparations.

This video demonstrates a dissection procedure for processing human pancreas into multiple storage formats. Anatomical orientation is maintained throughout the pancreatic regions to allow definition of regional islet composition and density.

人体胰腺的收集协议

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation ...

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low10; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22, far fewer exist for ICCs10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6. Although the examples presented here focus upon the pancreatic endoderm proliferation and &#946;&#160;cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

A protocol to isolate, culture, and image islet cell clusters (ICCs) derived from human fetal pancreatic cells is described.&#160; The method details the steps necessary to generate ICCs from tissue, culture as monolayers or in suspension as aggregates, and image for markers of proliferation and pancreatic cell fate decisions.

分离，培养和成像胎儿胰腺细胞集群

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely&#160;to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models.
Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method.
The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.

Current knowledge on the cellular basis of cardiac diseases mostly relies on studies on animal models. Here we describe and validate a novel method to obtain single viable cardiomyocytes from small surgical samples of human ventricular myocardium. Human ventricular myocytes can be used for electrophysiological studies and drug testing.

隔离与人类心室肌细胞的功能研究从新鲜手术样品

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

癌症干细胞从人类前列腺癌样本隔离

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Rapid Fractionation and Isolation of Whole Blood Components in Samples Obtained from a Community-based Setting

Collection and processing of whole blood samples in a non-clinical setting offers a unique opportunity to evaluate community-dwelling individuals both with and without preexisting conditions. Rapid processing of these samples is essential to avoid degradation of key cellular components. Included here are methods for simultaneous peripheral blood mononuclear cell (PBMC), DNA, RNA and serum isolation from a single blood draw performed in the homes of consenting participants across a metropolitan area, with processing initiated within 2 hr of collection. We have used these techniques to process over 1,600 blood specimens yielding consistent, high quality material, which has subsequently been used in successful DNA methylation, genotyping, gene expression and flow cytometry analyses. Some of the methods employed are standard; however, when combined in the described manner, they enable efficient processing of samples from participants of population- and/or community-based studies who would not normally be evaluated in a clinical setting. Therefore, this protocol has the potential to obtain samples (and subsequently data) that are more representative of the general population.

We outline a methodology for the processing of whole blood to obtain a variety of components for further analysis. We have optimized a streamlined protocol that enables rapid, high-throughput simultaneous processing of whole blood samples in a non-clinical setting.

快速分馏和全血的成分的分离样品中从社区为基础的设置获得

Collection and processing of whole blood samples in a non-clinical setting offers a unique opportunity to evaluate community-dwelling individuals both ...

Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic uses.
Exosomes have recently been shown to contain microRNAs (miRs). MiRs themselves have arisen as valuable biomarkers for diagnostic purposes. As specimen collection in clinics and hospitals is quite variable, miRNA isolation from whole bile varies substantially. To achieve robust, accurate and reproducible miRNA profiles from collected bile samples in a simple manner required the development of a high-quality protocol to isolate and characterize exosomes from bile. The method requires several centrifugations and a filtration step with a final ultracentrifugation step to pellet the isolated exosomes. Electron microscopy, Western blots, flow cytometry and multi-parameter nanoparticle optical analysis, where available, are crucial characterization steps to validate the quality of the exosomes. For the isolation of miRNA from these exosomes, spiking the lysate with a non-specific, synthetic miRNA from a species like Caenorhabditis elegans, i.e., Cel-miR-39, is important for normalization of RNA extraction efficiency. The isolation of exosome from bile fluid following this method allows the successful miRNA profiling from bile samples stored for several years at -80 &#176;C.

Bile fluid is a valuable source of extracellular vesicles/exosomes that contain potentially important biomarkers. This protocol represents a robust method to isolate exosomes from human bile for further analyses including miRNA profiling.

分离和剖析人力胆汁含外来体的MicroRNA-

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic ...

Simultaneous Flow Cytometric Characterization of Multiple Cell Types Retrieved from Mouse Brain/Spinal Cord Through Different Homogenization Methods

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central nervous system (CNS). However, further optimization of these technologies would benefit from methods allowing rapid and streamline determination of the extent of CNS and cell-specific targeting upon administration of viral vectors or nanoparticles in the body. Here, we present a protocol that takes advantage of the high throughput and multiplexing capabilities of flow cytometry to allow a straightforward quantification of different cell subtypes isolated from mouse brain or spinal cord, namely microglia/macrophages, lymphocytes, astrocytes, oligodendrocytes, neurons and endothelial cells. We apply this approach to highlight critical differences between two tissue homogenization methods in terms of cell yield, viability and composition. This could instruct the user to choose the best method depending on the cell type(s) of interest and the specific application. This method is not suited for analysis of anatomical distribution, since the tissue is homogenized to generate a single-cell suspension. However, it allows to work with viable cells and it can be combined with cell-sorting, opening the way for several applications that could expand the repertoire of tools in the hands of the neuroscientist, ranging from establishment of primary cultures derived from pure cell populations, to gene-expression analyses and biochemical or functional assays on well-defined cell subtypes in the context of neurodegenerative diseases, upon pharmacological treatment or gene therapy.

We present a flow cytometry method to identify simultaneously different cell types retrieved from mouse brain or spinal cord. This method could be exploited to isolate or characterize pure cell populations in neurodegenerative diseases or to quantify the extent of cell targeting upon in vivo administration of viral vectors or nanoparticles.

通过不同的均质化方法从小鼠大脑/脊髓中检索到的多种细胞类型的同步流细胞测量特征

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central ...

Hemocompatibility Testing of Blood-Contacting Implants in a Flow Loop Model Mimicking Human Blood Flow

The growing use of medical devices (e.g., vascular grafts, stents, and cardiac catheters) for temporary or permanent purposes that remain in the body&#39;s circulatory system demands a reliable and multiparametric approach that evaluates the possible hematologic complications caused by these devices (i.e., activation and destruction of blood components). Comprehensive in vitro hemocompatibility testing of blood-contacting implants is the first step towards successful in vivo implementation. Therefore, extensive analysis according to the International Organization for Standardization 10993-4 (ISO 10993-4) is mandatory prior to clinical application. The presented flow loop describes a sensitive model to analyze the hemostatic performance of stents (in this case, neurovascular) and reveal adverse effects. The use of fresh human whole blood and gentle blood sampling are essential to avoid the preactivation of blood. The blood is perfused through a heparinized tubing containing the test specimen by using a peristaltic pump at a rate of 150 mL/min at 37 &#176;C for 60 min. Before and after perfusion, hematologic markers (i.e., blood cell count, hemoglobin, hematocrit, and plasmatic markers) indicating the activation of leukocytes (polymorphonuclear [PMN]-elastase), platelets (&#946;-thromboglobulin [&#946;-TG]), the coagulation system (thombin-antithrombin III [TAT]), and the complement cascade (SC5b-9) are analyzed. In conclusion, we present an essential and reliable model for extensive hemocompatibility testing of stents and other blood-contacting devices prior to clinical application.

This protocol describes a comprehensive hemocompatibility evaluation of blood-contacting devices using laser-cut neurovascular implants. A flow loop model with fresh, heparinized human blood is applied to mimic blood flow. After perfusion, various hematologic markers are analyzed and compared to the values gained directly after blood collection for hemocompatibility evaluation of the tested devices.

流循环模型中血液接触植入物的血液相容性测试模拟人类血流

The growing use of medical devices (e.g., vascular grafts, stents, and cardiac catheters) for temporary or permanent purposes that remain in the ...

Validated LC-MS/MS Panel for Quantifying 11 Drug-Resistant TB Medications in Small Hair Samples

Drug resistant-tuberculosis (DR-TB) is a growing public health threat, and assessment of therapeutic drug levels may have important clinical benefits. Plasma drug levels are the current gold standard assessment, but require phlebotomy and a cold chain, and capture only very recent adherence. Our method uses hair, a matrix that is easily collected and reflective of long-term adherence, to test for 11 anti-TB medications. Previous work by our group shows that antiretroviral drug levels in hair are associated with HIV outcomes. Our method for DR-TB drugs uses 2 mg of hair (3 cm proximal to the root), which is pulverized and extracted in methanol. Samples are analyzed with a single LC-MS/MS method, quantifying 11 drugs in a 16 min run. Lower limits of quantification (LLOQs) for the 11 drugs range from 0.01 ng/mg to 1 ng/mg. Drug presence is confirmed by comparing ratios of two mass spectrometry transitions. Samples are quantified using the area ratio of the drug to the deuterated, 15N-, or 13C-labeled drug isotopologue. We used a calibration curve ranging from 0.001-100 ng/mg. Application of the method to a convenience sample of hair samples collected from DR-TB patients on directly observed therapy (DOT) indicated drug levels in hair within the linear dynamic range of nine of the eleven drugs (isoniazid, pyrazinamide, ethambutol, linezolid, levofloxacin, moxifloxacin, clofazimine, bedaquiline, pretomanid). No patient was on prothionamide, and the measured levels for ethionamide were close to its LLOQ (with further work instead examining the suitability of ethionamide&#8217;s metabolite for monitoring exposure). In summary, we describe the development of a multi-analyte panel for DR-TB drugs in hair as a technique for therapeutic drug monitoring during drug-resistant TB treatment.

Current methods of analyzing patients&#8217; adherence to complex drug resistant-tuberculosis (DR-TB) regimens can be inaccurate and resource-intensive. Our method analyzes hair, an easily collected and stored matrix, for concentrations of 11 DR-TB medications. Using LC-MS/MS, we can determine sub-nanogram drug levels that can be utilized to better understand drug adherence.

经过验证的LC-MS/MS面板，用于在小头发样本中量化11种耐药结核病药物

Drug resistant-tuberculosis (DR-TB) is a growing public health threat, and assessment of therapeutic drug levels may have important clinical benefits. ...