The authors thank Adi Sasson for the technical assistance and Helen Guez for the critical reading of the manuscript. This work was supported by European Research Council starting grant no. 336204.

实验按照委员会关于动物研究在特拉维夫大学批准的方案进行。 1.从小鼠胰腺组织隔离<ol><li> 对于成年小鼠: <ol><li>制备消化缓冲液:0.4毫克/毫升胶原酶P和0.1毫微克/毫升DNA酶I溶解在Hanks&#39;平衡盐溶液（HBSS）中。刚准备5毫升每只小鼠缓冲区的15毫升锥形管，并保持他们在冰上直到使用。 </li><li>根据机构指引安乐死的小鼠。 </li><li>解剖每只小鼠以去除胰腺（为说明，请参见参考文献18）;将其放置在含有HBSS培养皿。有可能在立体显微镜下执行此阶段，并且提高消化效率，切割组织分成2 - 4件。 </li><li>放置胰腺组织成含有消化缓冲液（在步骤1.1.1的方法制备）的管。保持冰管，直到所有的老鼠解剖和胰腺组织集。 </li><li>重复步骤1.1.2 - 1.1.4与剩余的小鼠。 </li></ol></li><li> 对于胚胎和新生小鼠: <ol><li>准备消化缓冲液:0.4毫克/毫升胶原酶P和0.1毫微克/毫升DNA酶I溶解在HBSS。在一个15毫升的锥形管2毫升每只小鼠缓冲 - 在出生后第7天（P7）或更小的胚胎和幼崽，刚准备1.5。在一个15毫升的锥形管4毫升，每鼠缓冲区 - 为P7以上的幼崽，准备3。置于冰上直至使用。 </li><li>根据机构指引安乐死的小鼠。对于胚胎，从母亲的子宫中删除所有的胚胎，并放置在含有PBS 10毫米的培养皿。在一个时间解剖一个胚胎。 </li><li>莱在其后面的鼠标，其头部指向从调查的路程。用细镊子，拉起腹部皮肤和开放的中线暴露腹腔到鼠标隔膜18。 </li><li>用细镊子，分离肝脏FR嗡背面腹壁。随着朝鼠尾连续运动，挖出内脏（包括肝，胃，脾，肠，肾和胰腺），并放置在含有HBSS培养皿。 </li><li>下一个立体显微镜，取出肝脏和肾脏，以暴露胰腺（ 图1）。用细镊子，从胃，十二指肠分离胰腺，最后，从脾。 </li><li>放置胰腺组织成含有消化缓冲液（在步骤1.2.1的方法制备）的管。对于P14以上的幼崽，切割组织成2块，增加消化效率。保持冰管，直到所有的老鼠解剖，并收集胰腺组织。 </li><li>重复步骤1.2.2 - 1.2.6与剩余的小鼠。 </li></ol></li></ol> 2.胰腺消化<ol><li>孵育在加热块含有胰腺组织（在消化缓冲液中）的试管，在37℃与agitat 30分钟离子在700rpm。 15分钟后，手动颠倒他们3撼动管 - 4倍，并把它们放回加热块。 注意:如果使用加热模块，无需搅拌功能，反转管每隔5 - 10分钟。确保组织被适当消化。如果不是这样，切割组织切成小块孵育前或增加搅动。 </li><li>停止组织消化，将管在冰上，加入10毫升冷HBSS的每个管。离心管，在4℃和300×g离心5分钟，吸出上清液。 </li><li> 再悬浮: <ol><li>对成年小鼠和P14或老年人幼仔，重新暂停与6毫升的PBS将沉淀，并通过放置在新的15毫升锥形收集聚丙烯管的顶端70微米的细胞滤网过滤它。以确保最大细胞回收，洗原管与另外的6毫升的PBS，并通过细胞过滤到收集管应变它。 </li><li>对于胚胎和幼崽年轻Ť汉P14，重新悬浮用2毫升的PBS将沉淀，并通过放置在一个5毫升圆底聚苯乙烯收集管（FACS管）的顶部有35微米的细胞滤网过滤它。以确保最大细胞回收，洗原管用另外2毫升的PBS，并通过细胞过滤到收集管应变它。 </li></ol></li><li>离心管，在4℃和300×g离心5分钟，吸出上清液。小心除去液体，如细胞松弛地附着在聚苯乙烯管中。 </li></ol> 3.制备标记单细胞<ol><li>缓冲制剂:对于通过FACS细胞分离，制备混合的PBS（不含氯化钙和氯化镁），5％牛胎儿血清（FBS）和5mM EDTA的排序缓冲器。对于流式细胞仪分析，通过用0.05％的叠氮化钠补充排序缓冲器准备分析缓冲液中。两个缓冲器可以存储在4-8℃下长达2个月。 </li><li>重新暂停排序或ANA细胞裂解液。重新暂停从胚胎和幼仔相比于1mL P7年轻分离各胰脏，从P8和满月在1.5毫升之间幼仔，并且从小鼠中超过一个月老年人在3mL。通过放置在圆底聚苯乙烯试管（FACS管）的顶部有35微米的细胞滤网过滤细胞。 </li><li>用于染色的控制，取出50到从在先前步骤（总样品体积的不超过5％）制备的样品100-μL等分试样，将它们放入新FACS管;包括用于每个荧光管使用，包括DAPI和荧光蛋白，以及用于未染色的对照。 注意:当使用转基因小鼠与表达荧光蛋白的细胞，或当细胞数量是有限的，包括非转基因组织作为染色对照。如果表达荧光蛋白的细胞没有进一步染色使用，请继续执行步骤3.8。 </li><li>降速细胞在300×g离心5分钟，在4℃和吸出上清液。 </li><li>对于集团王，再暂停（辅以山羊IgG 1μL100μL整理或分析缓冲液）与封闭液的细胞孵育在冰上30分钟。 </li><li>染色细胞表面标记物，制备包含在每个样品100微升的体积所有期望的荧光团标记的抗体的混合物。准备在排序或分析缓冲2X抗体稀释液（ 即，稀释抗体，以获得双所需浓度;如果1的最终稀释度:200是期望的，稀释抗体1:100）。未经洗涤封闭溶液，添加100μL的混合物在样品细胞达到200微升的最终染色体积。孵育在冰上30-60分钟，并在黑暗中。 </li><li>到设定的分析或分类参数（参见步骤4.3和5.2），制备仅包含使用（染色对照）抗体的一个额外的管。在分选或分析缓冲液（如在步骤3.6中所述）制备2×抗体稀释液。确保包括一个SI对于使用的每种荧光团，以及未染色对照ngle染色控制稀释。未经洗涤封闭溶液，加入抗体混合物的细胞（在步骤3.3中制备）来实现的200微升最终染色体积100微升。孵育30 - 在冰上并在黑暗中60分钟。 </li><li>填充每个管与分析或分类缓冲器4毫升的最大体积洗。任选地，通过35微米的细胞过滤网重新菌株细胞（如在步骤3.2中所述）。 </li><li>离心在4°C 300 XG 5分钟，小心地取出上清。 </li><li>在分析或分类缓冲器重新悬浮的样品。重悬在500微升的胚胎中分离的细胞，从幼仔相比于1mL1个月年轻化，从1 - 旧3个月2毫升，并从比在3mL 3个月以上的小鼠。染色控制可以被重新悬浮于缓冲液300微升。 </li><li>添加200毫微克/ DAPI毫升到重悬浮的细胞，以确定死亡细胞。确保包括管合作ntaining未染色细胞无DAPI的流式细胞仪的设置。进行到细胞分选（步骤4）或分析（步骤5）。 </li></ol> 4.细胞分选<ol><li> 准备工作: <ol><li>细胞用RNase排序之前RNA提取，涂层的1.5ml收集管抑制剂立即排序之前。为此，加入1毫升排序缓冲器，含有0.01 U / ml的RNA酶抑制剂，以无菌的1.5毫升收集管中。 5分钟后，涡管和除去液体。 </li><li>用于细胞之前细胞培养物分选，添加无菌培养介质的3毫升（Dulbecco氏改良的Eagle培养基（DMEM），补充有20％FBS，1％L-谷氨酰胺和青霉素 - 链霉素1％）到无菌的15毫升收集管。 </li></ol></li><li>装载管进入在FACS分拣机之前，涡旋它简单地重悬浮细胞。置于冰上剩余的管子。 </li><li>通过分析染色的控制，以确定排序参数（ 例如启动， </em&gt;电压和补偿）和分配门（ 例如，总的细胞群体，住DAPI阴性的细胞，细胞群进行排序）。 </li><li>一旦分拣参数和大门都设置了，装载样品并启动细胞分选入收集管。 注:排序条件高度依赖于仪器。我们使用100μm的喷嘴宽度，23.1 psi的压力，和5的最大排序速度。 </li><li>继续RNA提取或排序细胞的培养。 注:对于RNA提取，离心细胞以2000×g离心5分钟，并用一个标准提取协议继续之前除去多余的液体。用于培养细胞，如果细胞在非无菌条件下进行排序，由填充培养用培养基的管中并以尽量减少其污染培养前在300 xg离心离心它为7分钟洗两次。 </li></ol> 5.通过流式细胞仪细胞分析<ol><li> l在oading每个管进入细胞仪，涡流它简单地重悬浮细胞。置于冰上剩余的管子。 </li><li>通过以确定分析参数（ 例如，电压和补偿）分析未染色和单染色样品开始。 </li><li>一旦分析参数被设置，加载每个样品，包括染色控制，并记录结果。分析使用流式细胞分析软件所获得的结果。 </li></ol>

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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+Hedgehog+signaling+enhances+delivery+of+chemotherapy+in+a+mouse+model+of+pancreatic+cancer.">Inhibition of Hedgehog signaling enhances delivery of chemotherapy in a mouse model of pancreatic cancer.</a> Science. 324 (5933), 1457-1461 (2009).</li><li>Raz, Y., Erez, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+inflammatory+vicious+cycle:+Fibroblasts+and+immune+cell+recruitment+in+cancer.">An inflammatory vicious cycle: Fibroblasts and immune cell recruitment in cancer.</a> Experimental cell research. 319 (11), 1596-1603 (2013).</li></ol>

The authors have nothing to disclose.

在这里，我们描述了分离和分析胰腺微环境细胞的方法。这种方法可用于间充质细胞从胚胎和成年胰腺组织隔离。此外，我们成功地使用该协议来隔离从成人和新生儿胰腺5,17内皮细胞。然而，这可能不适合用于获得胰腺上皮细胞的可重复的单细胞悬浮液（可替代的协议在参考文献18中，23中描述，和24）。使用这种方法，荧光标记的细胞中，无论是表达荧光蛋白或免疫染色表面标记，可以通过FACS进行纯化或通过流式细胞仪分析。 RNA可以从纯化细胞提取介绍他们的基因表达模式。可选择地，纯化的细胞可以培养以建立用于随后的蛋白组学分析的细胞系。这种方法将使因素e的表征胰腺微环境，从而规范其器官，生理和病理生理学xpressed。 胰腺间叶细胞通过促进前体和分化的细胞5,9增殖支持组织器官。这些细胞被示出为支持人类胚胎干细胞的膨胀（人类胚胎干细胞）衍生的胰腺祖细胞17，25，26。因此，描绘的胚胎间充质因素的身份将促进目前的努力，以产生由hESCs和诱导多能干细胞（iPS细胞），为潜在的治疗糖尿病产生胰岛素的β细胞。小鼠遗传学研究允许的生长因子，如FGF10，由间质中产生的识别期间胰腺发育的早期阶段，以促进胰腺上皮膨胀3,9。与识别所述胚胎间充质表达的附加因素的目的，我们分离使用激光捕获显微切割这些细胞中，提取它们的RNA，并进行基因表达分析26。然而，除了是劳动密集的，这种方法依赖于识别基于其形态特征，其中上皮的分支进入周围间质（ 即，E12.5）之前限制了它的使用，以发育阶段的细胞。在后来的发展阶段的特征的间质细胞，我们采用这里5,17中记载的方法。 我们使用这种方法新生儿胰腺间质5，分析表面标志物的表达。此外，间充质细胞从Nkx3.2 -Cre的胚胎和新生儿胰组织中分离; R26-EYFP小鼠，是根据它们的荧光标记在这个鼠标线，并进行培养，建立细胞系17。这些细胞允许由胰腺间质与促进人类胚胎干细胞衍生的胰腺祖17的能力分泌因子识别的蛋白质组学分析。我们进一步用这种细胞的分离方法，以从RNA提取和基因表达分析17成人胰腺组织纯化的间充质细胞。因此，这种方法可以用于识别由胰腺间充质表达的基因和蛋白质，具有支持胰腺细胞发育的能力。 胰腺间充质细胞进一步显示出在胰腺肿瘤发生的作用。 PDAC的特征在于形成由成纤维细胞，免疫细胞的丰富成纤维结缔组织增生基质，和ECM 27。虽然基质被认为推动了许多发展类型的癌症中，显示抑制PDAC进展15，16，28。这表明，胰基质组分分泌抑制肿瘤发生的因素。此外，在间质细胞的组合物，以及在细胞表型的变化可以背后上的上皮细胞15，16，28的作用。因此这里描述的方法可以协助在表征不同类型的细胞，使一个PDAC基质相比，健康胰腺组织。这将进一步允许不同基质的细胞类型的纯化PDAC进展过程中表征其基因表达谱电位变化。然而，由于肿瘤27期间在胰腺癌的ECM组合物的变化，组织消化参数的调整，如纳入ADDIT的有理胶原酶类型或增加孵育时间，可能是需要的。

能量平衡和哺乳动物食物的消化依赖于适当的胰腺功能。成人胰腺由两个主要细胞区室的:外分泌和内分泌。外分泌细胞，包括产生和分泌消化酶和运输这些酶肠道管细胞，包括总胰1质量的80％以上的腺泡细胞。内分泌细胞，包括生产胰岛素的β细胞和胰高血糖素生产的α细胞中，在该嵌入外分泌组织和分泌的激素来调节血糖水平2胰岛被组织。 胰腺细胞通过一个高度调节，多步骤的过程3获得其分化命运。有证据表明，由神经元细胞，内皮细胞，和间质细胞提供外在线索引导在叔胰腺细胞的分化和增殖他胚胎3，4，5。一个例子是主动脉早期胰前体6的规范的要求。后来在发展，被示出的内皮细胞中都胰腺内分泌和外分泌细胞的发展中发挥中心作用，并促进β细胞分化4，6，7。被示出的间质细胞，以支持存活和共同胰祖细胞的扩增，主要是通过生长因子FGF10 8,9的分泌。我们已进一步显示，这些细胞支持内分泌和外分泌的前体在胚胎胰腺5的扩散，以及分化的细胞（包括腺泡和β细胞）。最近，间充质细胞还证明是调节内分泌细胞的分化10。 在成人中，β细胞功能和质量均表现依赖于细胞中的微环境，包括神经元细胞，免疫细胞和内皮细胞，以及周细胞11，12，13。在损伤，均表现内皮细胞招募免疫细胞到胰腺，促进β细胞复制13。内皮细胞进一步示出，以产生细胞外基质（ECM）组件以支持胰岛素表达和β细胞功能14。我们最近表现出胰岛周细胞对β细胞功能11的要求。最后，被示出的胰基质的细胞来调节胰腺导管腺癌（PDAC）15，16的进展。然而，标识指导胰腺发育，功能和肿瘤外在线索的实体，在很大程度上是未知。 识别由胰腺微环境的细胞提供线索需要表征由这些细胞表达的基因和蛋白质。这依赖于以和/或建立的细胞系进行基因表达和蛋白质组分析中分离从胰腺这些细胞。这里，我们提出了一种方法，通过利用表达荧光蛋白或免疫荧光标记的细胞或细胞组织的酶消化和荧光激活细胞分选（FACS）对胰腺微环境的间充质细胞分离。成功地执行该协议，以分离和分析黄色荧光蛋白（YFP）-expressing胚胎，新生儿和成人胰腺5，17间充质细胞。

<table border="0" cellpadding="0" cellspacing="0" width="766">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Collagenase P</td>
			<td style="width:167px;">Roche</td>
			<td style="width:191px;">11213865001</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNase</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td style="width:191px;">D5025-15KU</td>
			<td>The effective units should be at least 2000 unitz/ml protein</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hanks&#8217; Balanced Salt solution (HBSS)</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td style="width:191px;">H6648</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:167px;">Biological Industries</td>
			<td style="width:191px;">04-127-1A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">EDTA</td>
			<td style="width:167px;">Biological Industries</td>
			<td style="width:191px;">01-862-1A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Azide</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td style="width:191px;">S2002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Goat IgG serum</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td style="width:191px;">G9023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DAPI</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td style="width:191px;">D9542</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNAse inhibitor</td>
			<td style="width:167px;">Invitrogen</td>
			<td style="width:191px;">N8080119</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td style="width:167px;">Invitrogen</td>
			<td style="width:191px;">11965092</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Glutamine</td>
			<td style="width:167px;">Biological Industries</td>
			<td style="width:191px;">03-020-1B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-streptomycin</td>
			<td style="width:167px;">Biological Industries</td>
			<td style="width:191px;">03-031-1B</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Dulbecco&#8217;s Phosphate Buffered Saline (PBS)</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td style="width:191px;">D8537</td>
			<td>Without Calcium Chloride and Magnesium Chloride</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;"></td>
			<td style="width:167px;"></td>
			<td style="width:191px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;"></td>
			<td style="width:167px;"></td>
			<td style="width:191px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">1.5 ml tubes</td>
			<td style="width:167px;">Sarstedt</td>
			<td style="width:191px;">72 690</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">15 ml conical tube</td>
			<td style="width:167px;">Corning</td>
			<td style="width:191px;">430052</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Round Bottom Polystyrene 5 ml tube&#160;</td>
			<td style="width:167px;">Corning</td>
			<td style="width:191px;">352008</td>
			<td>FACS tube&#39;. Make sure tube is compatible with the flow cytomter to be used, as there are slight differences in required tubes between brands</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5ml tube with 35 &#956;m cell strainer Snap Cap</td>
			<td style="width:167px;">Corning</td>
			<td style="width:191px;">352235</td>
			<td>FACS tube&#39;&#160;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70 &#956;m cell strainer&#160;</td>
			<td style="width:167px;">Miltenyi Biotec</td>
			<td style="width:191px;">130-098-462</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;"></td>
			<td style="width:167px;"></td>
			<td style="width:191px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Heating block with agitation&#160;</td>
			<td style="width:167px;">Eppendorf</td>
			<td style="width:191px;">ThermoMixer C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Centrifuge</td>
			<td style="width:167px;">ThermoFisher</td>
			<td style="width:191px;">Heraeus Megafuge 40R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Steromicroscope</td>
			<td style="width:167px;">Nikon</td>
			<td style="width:191px;">SMZ 745</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Cell sorter</td>
			<td style="width:167px;">BD Biosciences</td>
			<td style="width:191px;">FACSAria IIu</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">flow cytometer</td>
			<td style="width:167px;">Beckman Coulter</td>
			<td style="width:191px;">Gallios&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolating and analyzing cells of the pancreas mesenchyme by flow cytometry

The pancreas is comprised of epithelial cells that are required for food digestion and blood glucose regulation. Cells of the pancreas microenvironment, including endothelial, neuronal, and mesenchymal cells were shown to regulate cell differentiation and proliferation in the embryonic pancreas. In the adult, the function and mass of insulin-producing cells were shown to depend on cells in their microenvironment, including pericyte, immune, endothelial, and neuronal cells. Lastly, changes in the pancreas microenvironment were shown to regulate pancreas tumorigenesis. However, the cues underlying these processes are not fully defined. Therefore, characterizing the different cell types that comprise the pancreas microenvironment and profiling their gene expression are crucial to delineate the tissue development and function under normal and diseased states. Here, we describe a method that allows for the isolation of mesenchymal cells from the pancreas of embryonic, neonatal, and adult mice. This method utilizes the enzymatic digestion of mouse pancreatic tissue and the subsequent fluorescence-activated cell sorting (FACS) or flow-cytometric analysis of labeled cells. Cells can be labeled by either immunostaining for surface markers or by the expression of fluorescent proteins. Cell isolation can facilitate the characterization of genes and proteins expressed in cells of the pancreas mesenchyme. This protocol was successful in isolating and culturing highly enriched mesenchymal cell populations from the embryonic, neonatal, and adult mouse pancreas.

胰腺间质的发展和成年期间需要。这里所描述的方法允许间质细胞从胚胎，新生儿和成人胰腺的隔离。间充质细胞，但没有其他类型的细胞，表达Nkx3.2 -Cre的胰腺黄色荧光蛋白（YFP）; R26R -YFP小鼠5，11，17，19。在开发期间，Nkx3.2（也称为BapX1）由胚胎胰腺，胃和肠间质，以及在骨架体节19，20，21的一个子集表示。该基因在胰腺间充质中表达从E9.5至E11.5，允许来自E9.5 5 Nkx3.2 -Cre的控制下的基因表达， </suP&gt; 19，20。基于此标记，细胞可从大容量的胰腺组织用流式细胞术纯化。 图2显示了从胚胎，新生儿和成人胰腺组织，分离为这里所描述的单细胞的流式细胞仪分析。而与非转基因胰腺组织不含有荧光细胞，Nkx3.2 -Cre; R26R -YFP从所有分析的年龄胰腺组织含有不同的YFP标记的细胞群体（ 图2;标有门）。 下面这里描述的方法中，表达荧光蛋白的细胞可以是纯化的或通过流式细胞仪分析，有或没有附加的免疫染色。例如，根据荧光标记排序后，间质细胞可以培养建立的细胞系（至少5代）， 如图3A所示 。注ŧ<html>他fibrocytic培养的细胞，典型到间充质细胞的形态。此外，使用该系统由分选的细胞来分析基因表达。为此，RNA从排序的间充质细胞中提取合成cDNA和基因的表达水平通过qPCR分析。这样的分析表明，有序细胞表达泛间充质标记物波形蛋白（ 图3B）。最后，由胰腺细胞表面标记物表达可通过流式细胞术进行分析。例如，我们分离从Nkx3.2 -Cre的胰腺组织细胞; R26R -YFP使用此处描述和与细胞表面糖蛋白CD9，将其报告给由成纤维细胞22表示染色它们的方法成年小鼠。如在图3C中所示，所有的荧光标记的Nkx3.2 -Cre细胞; R26R -YFP胰腺表达CD9。 tp_upload / 55344 / 55344fig1.jpg"/&gt; 图1:胚胎和新生儿的胰腺组织。隔离整个胃肠道，包括胃，脾，肠，胰腺，一个E15.5胚胎（A）和P4小狗（B）的。胰腺组织划定了蓝线。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55344/55344fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/55344/55344fig2.jpg" /> 图2:间质细胞荧光标记的Nkx3.2 -Cre; R26R -YFP胰腺组织。从非转基因（左图）和Nkx3.2 -Cre分离胰腺细胞的流式细胞仪数据; R26R -YFP在不同年龄的转基因（右图）小鼠:胚胎（A），新生儿（B），和广告ULT（C）。分析细胞的侧向散射（y轴）和黄色荧光（x轴）。盖茨（标有"D"）指示在转基因小鼠中的YFP +细胞群的存在，但不是在非转基因对照。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55344/55344fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/55344/55344fig3.jpg" /> 图3:孤立的胰腺细胞分析。从Nkx3.2 -Cre的胰腺组织排序（A）细胞; R26R -YFP（如在图2B中所述）新生小鼠中培养，建立的细胞系。培养的细胞进行成像荧光（绿色;右和左小图）和相位对比（灰色;右图）。 （B）显示Vimentin1（Vim1）棒图表达水平通过YFP + -sorted细胞Nkx3.2 -Cre的胰腺组织; R26R -YFP成年小鼠（如在图1C中描述的;绿色）相比，未分选的胰腺组织（黑）。提取RNA，并通过定量PCR分析基因表达;表达标准化为环素 。 N = 4，*** P &lt;0.001。数据代表平均值±SD。 （C）从Nkx3.2 -Cre分散胰腺细胞的流式细胞仪数据; R26R -YFP成年小鼠免疫染色别藻蓝蛋白（APC）缀合的抗CD9抗体和分析的APC（y轴）和黄色荧光（x轴）。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55344/55344fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Isolating and Analyzing Cells of the Pancreas Mesenchyme by Flow Cytometry at JoVE.com

Isolating and Analyzing Cells of the Pancreas Mesenchyme by Flow Cytometry

这里，我们描述了一种用于细胞在胰腺微环境从胚胎，新生儿和成年小鼠组织的隔离，着眼于间充质细胞的分离。此方法允许细胞基因表达和蛋白质分泌的分析以阐明调节胰腺发育，功能，和肿瘤发生的外在信号。

隔离并通过流式细胞仪分析胰腺间充质细胞

The pancreas is comprised of epithelial cells that are required for food digestion and blood glucose regulation. Cells of the pancreas microenvironment, ...

isolating-analyzing-cells-pancreas-mesenchyme-flow

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

12.1K 次观看.  Sackler Faculty of Medicine, Tel Aviv University. 这里，我们描述了一种用于细胞在胰腺微环境从胚胎，新生儿和成年小鼠组织的隔离，着眼于间充质细胞的分离。此方法允许细胞基因表达和蛋白质分泌的分析以阐明调节胰腺发育，功能，和肿瘤发生的外在信号。 

视频: Isolating and Analyzing Cells of the Pancreas Mesenchyme by Flow Cytometry

Step-specific Sorting of Mouse Spermatids by Flow Cytometry

The differentiation of mouse spermatids is one critical process for the production of a functional male gamete with an intact genome to be transmitted to the next generation. So far, molecular studies of this morphological transition have been hampered by the lack of a method allowing adequate separation of these important steps of spermatid differentiation for subsequent analyses. Earlier attempts at proper gating of these cells using flow cytometry may have been difficult because of a peculiar increase in DNA fluorescence in spermatids undergoing chromatin remodeling. Based on this observation, we provide details of a simple flow cytometry scheme, allowing reproducible purification of four populations of mouse spermatids fixed with ethanol, each representing a different state in the nuclear remodeling process. Population enrichment is confirmed using step-specific markers and morphological criterions. The purified spermatids can be used for genomic and proteomic analyses.

We describe a sorting strategy for mouse spermatids using flow cytometry. Spermatids are sorted into four highly pure populations, including round (spermiogenesis steps 1-9), early elongating (spermiogenesis steps 10-12), late elongating (spermiogenesis steps 13-14) and elongated spermatids (spermiogenesis steps 15-16). DNA staining, size and granulosity are used as selection parameters.

步特定老鼠的精子细胞分选流式细胞仪

The differentiation of mouse spermatids is one critical process for the production of a functional male gamete with an intact genome to be transmitted to ...

科研

发育生物学

Isolating Hair Follicle Stem Cells and Epidermal Keratinocytes from Dorsal Mouse Skin

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by distinct pools of SCs, which are located in the permanent portion of the HF, a region known as the bulge. These multipotent bulge SCs were initially identified as slow cycling label retaining cells; however, their isolation has been made feasible after identification of specific cell markers, such as CD34 and keratin 15 (K15). Here, we describe a robust method for isolating bulge SCs and epidermal keratinocytes from mouse HFs utilizing fluorescence activated cell-sorting (FACS) technology. Isolated hair follicle SCs (HFSCs) can be utilized in various in vivo grafting models and are a valuable in vitro model for studying the mechanisms that govern multipotency, quiescence and activation.

An ideal model for studying adult stem cell biology is the mouse hair follicle. Here we present a protocol for isolating different populations of hair follicles stem cells and epidermal keratinocytes, employing enzymatic digestion of mouse dorsal skin followed by FACS analysis.

从背小鼠皮肤分离毛囊干细胞和表皮角质形成细胞

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by ...

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. However, it is still unknown whether stem and progenitor cells exist in the adult pancreas. Research strategies using cre-lox lineage-tracing in adult mice have yielded results that either support or refute the idea that beta cells can be generated from the ducts, the presumed location where adult pancreatic progenitors may reside. These in vivo cre-lox lineage-tracing methods, however, cannot answer the questions of self-renewal and multi-lineage differentiation-two criteria necessary to define a stem cell. To begin addressing this technical gap, we devised 3-dimensional colony assays for pancreatic progenitors. Soon after our initial publication, other laboratories independently developed a similar, but not identical, method called the organoid assay. Compared to the organoid assay, our method employs methylcellulose, which forms viscous solutions that allow the inclusion of extracellular matrix proteins at low concentrations. The methylcellulose-containing assays permit easier detection and analyses of progenitor cells at the single-cell level, which are critical when progenitors constitute a small sub-population, as is the case for many adult organ stem cells. Together, results from several laboratories demonstrate in vitro self-renewal and multi-lineage differentiation of pancreatic progenitor-like cells from mice. The current protocols describe two methylcellulose-based colony assays to characterize mouse pancreatic progenitors; one contains a commercial preparation of murine extracellular matrix proteins and the other an artificial extracellular matrix protein known as a laminin hydrogel. The techniques shown here are 1) dissociation of the pancreas and sorting of CD133+Sox9/EGFP+ ductal cells from adult mice, 2) single cell manipulation of the sorted cells, 3) single colony analyses using microfluidic qRT-PCR and whole-mount immunostaining, and 4) dissociation of primary colonies into single-cell suspensions and re-plating into secondary colony assays to assess self-renewal or differentiation.

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

In vitro colony assays to detect self-renewal and differentiation of progenitor cells isolated from adult murine pancreas are devised. In these assays, pancreatic progenitors give rise to cell colonies in 3-dimensional space in methylcellulose-containing semi-solid medium. Protocols for handling single cells and characterization of individual colonies are described.

体外测定菌落为表征的三效祖细胞从成人胰腺小鼠隔离

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. ...

Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where they act as front line sentinels of infection and tissue damage. While other immune cells are continuously renewed from hematopoietic stem and progenitor cells (HSPC) located in the bone marrow, a lineage of macrophages, known as resident macrophages, have been shown to be self-maintained in tissues without input from bone marrow HSPCs. This lineage is exemplified by microglia in the brain, Kupffer cells in the liver and Langerhans cells in the epidermis among others. The intestinal and colon lamina propria are the only adult tissues devoid of HSPC-independent resident macrophages. Recent investigations have identified that resident macrophages originate from the extra-embryonic yolk sac hematopoiesis from progenitor(s) distinct from fetal hematopoietic stem cells (HSC). Among yolk sac definitive hematopoiesis, erythromyeloid progenitors (EMP) give rise both to erythroid and myeloid cells, in particular resident macrophages. EMP are only generated within the yolk sac between E8.5 and E10.5 days of development and they migrate to the fetal liver as early as circulation is connected, where they expand and differentiate until at least E16.5. Their progeny includes erythrocytes, macrophages, neutrophils and mast cells but only EMP-derived macrophages persist until adulthood in tissues. The transient nature of EMP emergence and the temporal overlap with HSC generation renders the analysis of these progenitors difficult. We have established a tamoxifen-inducible fate mapping protocol based on expression of the macrophage cytokine receptor Csf1r promoter to characterize EMP and EMP-derived cells in vivo by flow cytometry.

While infiltrating macrophages are continuously recruited to adult tissues from circulating precursors, resident macrophages seed their tissue during development, where they are maintained without further input from progenitors. The progenitors for resident macrophages were recently identified. Here, we present methods for the genetic fate mapping of the resident macrophage progenitors.

通过流式细胞术鉴定小鼠胚胎中的红细胞生成素及其后代

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where ...

A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. Currently, it allows the discrimination of up to 9 murine germ cell populations with high yield and purity. This high-resolution in discrimination and purification is possible due to unique changes in chromatin structure and quantity throughout spermatogenesis. These patterns can be captured by flow cytometry of male germ cells stained with fluorescent DNA-binding dyes such as Hoechst-33342 (Hoechst). Herein is a detailed description of a recently developed protocol to isolate mammalian testicular germ cells. Briefly, single cell suspensions are generated from testicular tissue by mechanical dissociation, double stained with Hoechst and propidium iodide (PI) and processed by flow cytometry. A serial gating strategy, including the selection of live cells (PI negative) with different DNA content (Hoechst intensity), is used during FACS sorting to discriminate up to 5 germ cell types. These include, with corresponding average purities (determined by microscopy evaluation): spermatogonia (66%), primary (71%) and secondary (85%) spermatocytes, and spermatids (90%), further separated into round (93%) and elongating (87%) subpopulations. Execution of the entire workflow is straightforward, allows the isolation of 4 cell types simultaneously with the appropriate FACS machine, and can be performed in less than 2 h. As reduced processing time is crucial to preserve the physiology of ex vivo cells, this method is ideal for downstream high-throughput studies of male germ cell biology. Moreover, a standardized protocol for multispecies purification of mammalian germ cells eliminates methodological sources of variables and allows a single set of reagents to be used for different animal models.

This work describes the standardization of a method to obtain purified germ cell populations from testicular tissue of different mammalian species. It is a straightforward protocol that combines mechanical testis dissociation, staining with Hoechst-33342 and propidium iodide, and FACS sorting, with wide applications in comparative studies of male reproductive biology.

通过机械组织分离和流式细胞术对哺乳动物雄性生殖细胞多特异性纯化的标准化方法

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. ...

Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, antibodies for specific cell markers are applied on tissue sections. In adult skeletal muscle, satellite cells (SCs) are stem cells that contribute to muscle repair and regeneration. Therefore, it is important to visualize and trace the satellite cell population under different physiological conditions. In resting skeletal muscle, SCs reside between the basal lamina and myofiber plasma membrane. A commonly used marker for identifying SCs on the myofibers or in cell culture is the paired box protein Pax7. In this article, an optimized Pax7 immunofluorescence protocol on skeletal muscle sections is presented that minimizes non-specific staining and background. Another antibody that recognizes a protein (laminin) of the basal lamina was also added to help identify SCs. Similar protocols can also be used to perform double or triple labeling with Pax7 and antibodies for additional proteins of interest.

The precise identification of satellite cells is essential for studying their functions under various physiological and pathological conditions. This article presents a protocol to identify satellite cells on adult skeletal muscle sections by immunofluorescence-based staining.

Pax7 和层粘连蛋白抗体免疫荧光法鉴定骨骼肌卫星细胞

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

秀丽线虫生殖的计算分析研究细胞核、蛋白质和细胞骨架的分布

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Use of Hematopoietic Stem Cell Transplantation to Assess the Origin of Myelodysplastic Syndrome

Myelodysplastic syndromes (MDS) are a diverse group of hematopoietic stem cell disorders that are defined by ineffective hematopoiesis, peripheral blood cytopenias, dysplasia, and a propensity for transformation to acute leukemia. NUP98-HOXD13 (NHD13) transgenic mice recapitulate human MDS in terms of peripheral blood cytopenias, dysplasia, and transformation to acute leukemia. We previously demonstrated that MDS could be transferred from a genetically engineered mouse with MDS to wild-type recipients by transplanting MDS bone marrow nucleated cells (BMNC). To more clearly understand the MDS cell of origin, we have developed approaches to transplant specific, immunophenotypically defined hematopoietic subsets. In this article, we describe the process of isolating and transplanting specific populations of hematopoietic stem and progenitor cells. Following transplantation, we describe approaches to assess the efficiency of transplantation and persistence of the donor MDS cells.

We describe the use of hematopoietic stem cell transplantation (HSCT) to assess the malignant potential of genetically engineered hematopoietic cells. HSCT is useful for evaluating various malignant hematopoietic cells in vivo as well as generating a large cohort of mice with myelodysplastic syndromes (MDS) or leukemia to evaluate novel therapies.

应用造血干细胞移植评估骨髓增生异常综合征的起源

Myelodysplastic syndromes (MDS) are a diverse group of hematopoietic stem cell disorders that are defined by ineffective hematopoiesis, peripheral blood ...

Real-time Live-cell Flow Cytometry to Investigate Calcium Influx, Pore Formation, and Phagocytosis by P2X7 Receptors in Adult Neural Progenitor Cells

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a method whereby live-cell flow cytometry is extended upon to analyze the multiple functions of P2X7 receptor activation in real-time. Using a time module installed on a flow cytometer, live-cell functionality can be assessed and plotted over a given time period to explore the kinetics of calcium influx, transmembrane pore formation, and phagocytosis. This simple method is advantageous as all three canonical functions of the P2X7 receptor can be assessed using one machine, and the gathered data plotted over time provides information on the entire live-cell population rather than single-cell recordings often obtained using technically challenging patch-clamp methods. Calcium influx experiments use a calcium indicator dye, while P2X7 pore formation assays rely on ethidium bromide being allowed to pass through the transmembrane pore formed upon high agonist concentrations. Yellow-green (YG) latex beads are utilized to measure phagocytosis. Specific agonists and antagonists are applied to investigate the effects of P2X7 receptor activity. Individually, these methods can be modified to provide quantitative data on any number of calcium channels and purinergic and scavenger receptors. Taken together, they highlight how the use of real-time live-cell flow cytometry is a rapid, cost-effective, reproducible, and quantifiable method to investigate P2X7 receptor function.

Providing single-cell sensitivity, real-time flow cytometry is uniquely suited to quantify multimodal receptor functions of live cultures. Using adult neural progenitor cells, the P2X7 receptor function was assessed via calcium influx detected by calcium indicator dye, transmembrane pore formation by ethidium bromide uptake, and phagocytosis using fluorescent latex beads.

P2X7 受体实时活细胞流式细胞仪检测成人神经祖细胞中的钙影响、毛孔形成和吞噬作用

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

人诱导多能干细胞体外生成体细胞衍生物的研究

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...