This work was supported by grant from NIDDK (DK065949 for GG). We thank Dr. Brenda M. Jarvis and Jeff Duryea Jr. for reading the manuscript.

动物的使用遵循协议中号规定的程序/一百八十一分之十一由范德比尔特机构动物护理和使用委员会批准谷。 CD1或CBA / BL6小鼠从商业供应商处购买和交叉在范德比尔特动物设施获得新生小鼠。 1.小鼠制备，股票解决方案和设备<ol><li>对于鼠标交叉:成立一个跨鼠标并记录日期堵漏帮助实验计划。 CD1小鼠通常交配后给周围19天诞生。使用CD1或CBA / BL6穿越获得CD1或CBA / BL6新生儿，分别。两条线之间Intercrosses利用CD1雌性和CBA / BL6男性。 </li><li>对于胶原酶股票:体重200毫克IV型胶原酶具有高精确度的平衡。转移至50毫升离心管中。溶解在40ml的HBSS用的 Ca 2+ / Mg 2+离子 （1.26和0.5mM，分别地）做出5毫克/毫升贮备液。 <ol><li>允许胶原酶溶解于轻轻摇动约30分钟。分装并保存在-20℃的股票，这对于至少一年保持有效冻结的解决方案。 </li></ol></li><li>高压灭菌消毒几双镊子，剪刀和P20技巧解剖和胰岛采摘。 </li><li>对于1M的葡萄糖股票:称取9克葡萄糖50毫升离心管中，加入50毫升RPMI 1640媒体溶解葡萄糖。保持在4℃直至使用，但不超过6个月。 </li></ol> 2.工作方案注:胰岛分离的日子，准备以下试剂。 <ol><li>对于IV型胶原酶工作液:解冻，并稀释冰上胶原酶股票。免除0.15毫升股票进入各1.7毫升离心管。添加1.35毫升HBSS与钙 /镁离子 。倒置该管5次。留在冰上15分钟。 <ol><li>旋转以高速在微量3分钟。转移上清液至新管。留在冰上，直到使用。丢弃不溶性碎片管。 </li></ol></li><li>对于完全RPMI 1640培养基:添加2.5毫升的1摩尔葡萄糖，50毫升热灭活的FBS和5mL的100×青霉素 - 链霉素库存入500毫升的RPMI 1640培养基。留在冰上，直到使用。 </li></ol> 3.胰腺分离和消化<ol><li>安乐死用异氟烷新生儿，根据批准的方案随后断头。 </li><li>搁浅了，肚子朝上鼠标。用70％乙醇消毒喷雾。 </li><li>提起腹部皮肤与镊子和纵向切开皮肤和肌肉层以及一对剪刀的中线，从生殖器区域到肋笼。这将打开腹腔暴露所有的内脏。 </li><li>传输所有内脏100 cm的天线用一对镊子。定位胰腺，这是一个分散的器官与粘附到胃，脾和粘附到十二指肠11腹侧部的背部。 </LI&gt; <li>添加HBSS入菜淹没的器官。在溶液中，胰腺组织不再执着于十二指肠，胃或脾。这是因为其典型的白色下解剖范围容易识别。使用一对镊子的从周围组织和传送远离剥离胰腺组织到一个新的60mm培养皿用HBSS。 </li><li>切用剪刀每个胰腺成片比在任何尺寸为5mm小。转移到1.5毫升离心管中。多达五个胰腺（年龄小于P7）可以在一个管被消化。对于P8-P16的小鼠，使用每个胰腺一管。 </li><li>加0.5 - 1毫升胶原酶工作液到每个管（0.2毫升每个P1-P7胰腺使用0.5mL的每个P8-P16胰腺）。留在37℃培养箱管长达15分钟。反转管每分钟两次。 </li><li> 〜5分钟后，点击管监视胰腺组织的消化状态。继续消化过程，直到大部分胰腺组织表现为零散的细胞团，直径&lt;2毫米。裂解液出现浑浊，这是由于正常的腺泡细胞自溶。 </li></ol> 4.裂解液洗<ol><li>在微型旋转溶解物在500×g离心10秒。用P1,000的Pipetman去除上清层。上清应该出现浑浊，但缺乏细胞团。不使用抽吸，其可以迅速地吸走在底部的组织碎片由于一些粘DNA的存在。 </li><li>加入1毫升RPMI 1640完整的媒体。轻轻拍打管悬浮分散的裂解液。反转向上和向下10倍。重复步骤4.1。 </li><li>重复步骤4.2两次或者直到上清出现明显的。在1毫升完全RPMI 1640培养基重悬水洗胰腺片段。 </li></ol> 5.胰岛分离注:对于胰腺小的数字，直接用手采摘如下（5.1），可用于胰岛ISOL通货膨胀。对于大量的胰腺（&gt; 6），在5.2中列出的方法是首选。 <ol><li>直接手工采摘: <ol><li>从步骤4.3传输裂解液60毫米的菜。加入5毫升完全RPMI 1640媒体。在解剖显微镜下把。 </li><li>建立在显微镜明视场照明。调整光强度到最大功率的〜50％。在此条件下，胰岛显示为淡粉红色簇，而腺泡细胞为暗不规则形群集（ 图1A）。 〜50X（结合目标的能力和目镜）的放大倍数，建议提供可视化胰岛的最佳方法，同时将枪头开放采摘。 </li><li>拿起胰岛与P20的枪头，同时避免腺泡集群。分配胰岛富集馏分到一个新的60mm培养皿的RPMI完全培养基（ 图1B）。 </li><li>重复手工采摘过程中的两倍多。离开纯胰岛在新60毫米培养皿用4mL的完全RPMI培养基（ 图1C）。 </li></ol></li><li>梯度离心法: <ol><li>制备是在10.77克/毫升的密度预装用2mL多聚蔗糖和钠泛影15毫升离心管中。离开冰管，直到使用。 </li><li>传送来自步骤4.3的悬浮液到用巴氏吸管的多聚蔗糖和钠泛影葡胺溶液的顶部。请勿打扰底层。从多达10个新生或2 P17胰腺组织可以加载到每15毫升管中。 </li><li>旋在〜600 XG所述管中的摆动转子15分钟，在4℃。使用制动开关设定。 </li><li>离心后，转移媒体和相间层（请注意，理想的操作条件下，胰岛将位于离心后的多聚蔗糖和培养基之间的相间）到一个新的15毫升管中。加入10 mL媒体，轻轻但调匀 。 </li><li>降速胰岛浓缩部分在300×g离心5分钟。取出洗净媒体。重复洗一次完全RPMI媒体。悬浮颗粒在4毫升媒体，它主要含有胰岛和导管（ 图1D）。 </li><li>手工挑选胰岛如第5.1节所述。 </li></ol></li></ol> 6.化验GSIS在分离的胰岛<ol><li>孵育从步骤5.1.4在完全RPMI培养基的胰岛在37℃组织培养孵化2小时。 </li><li>使用移液管除去在解剖显微镜下的RPMI。添加3与2.8毫米的葡萄糖毫升KRB溶液到板3。摇动10次洗小岛。删除KRB解决方案。 <ol><li>重复洗涤过程中一次。孵育用3mL KRB溶液胰岛在37℃在培养箱中1小时。 </li></ol></li><li>等分试样1毫升KRB到12孔板的每个孔中。预暖KRB的股票和板块37℃的孵化器。 </li><li>与预热KRB洗两次胰岛按照6.2。横贯FER 10胰岛到预热板的各孔中。孵育在37℃培养箱。 </li><li> 45分钟后，撤上清50μL的。这包含在基础葡萄糖分泌胰岛素溶液。 </li><li>加入32微升500毫米的葡萄糖。摇动板20倍到该溶液中混合。孵育在37℃培养箱。摇动板5次每隔5分钟。 </li><li> 45分钟后，撤50微升上清液。这是高糖分泌胰岛素。 </li><li>传输所有胰岛1.5mL管。冻结在-20℃30分钟。解冻在室温下5分钟。重复冷冻和解冻过程。 </li><li>添加0.5mL的70％乙醇的20mM HCl中。留下过夜。这是留在小岛的胰岛素。 </li><li>用常规ELISA法测定胰岛素水平，先前3所述。 </li></ol>

<ol>
	<li>Matsumoto, S., 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=Improvement+of+pancreatic+islet+cell+isolation+for+transplantation.">Improvement of pancreatic islet cell isolation for transplantation.</a> Proc (Bayl Univ Med Cent). 20 (4), 357-362 (2007).</li><li>Zmuda, E. J., Powell, C. A., Hai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+murine+islet+isolation+and+subcapsular+kidney+transplantation.">A method for murine islet isolation and subcapsular kidney transplantation.</a> J Vis Exp. 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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Source Book of Enzymes, Source Book of Enzymes. , (1997).</li><li>Bock, T., Pakkenberg, B., Buschard, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+background+determines+the+size+and+structure+of+the+endocrine+pancreas.">Genetic background determines the size and structure of the endocrine pancreas.</a> Diabetes. 54 (1), 133-137 (2005).</li><li>Bouwens, L., Rooman, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+pancreatic+beta-cell+mass.">Regulation of pancreatic beta-cell mass.</a> Physiol Rev. 85 (4), 1255-1270 (2005).</li></ol>

The authors declare no competing financial interests for the described work.

在这里，我们提供胰岛分离的一步一步的协议，从是传统灌注太小胰腺。据预计产量胰岛准备好所有基于胰岛研究，如β细胞的纯化，基因表达分析，胰岛β细胞的成熟，增殖，细胞应激反应，细胞存活，代谢和功能GSIS维护等 。这将是，以我们所知，第一次详细目视协议，引导新的研究人员从新生小鼠进行胰岛分离的。如果没有这些视觉助手，广泛的试验和错误是预期的大多数研究人员实?...

Isolating pure pancreatic islets is essential for assaying glucose stimulated insulin secretion (GSIS) of beta cells and for islet transplantation from cadaveric donors 1-3. It is also necessary to establish endocrine gene expression in islet cells 4, 5. For this purpose, detailed protocols have been established to allow for isolation of pancreatic islets from large pancreata (6 and references therein). These methods are based on enzymatic perfusion to dissociate acinar from islet tissues, coupled with gradient separation and hand picking. Thus, islet isolation from large pancreas can be performed readily in most laboratories. On the other hand, no detailed step-by-step protocol exists to allow for the isolation of islets from pancreata that are too small to perfuse.
Studying gene expression and function of neonatal islets is important. Neonate islets have different properties from adults in insulin secretion and proliferation capability 7, 8. However, isolating islets from newly born animals, especially mice is challenging due to the small size of the newly born pancreas. The size prevents the usual perfusion process when collagenase is injected though the pancreatic duct. Indeed, several papers have presented studies along these lines, with enzyme or non-enzyme aided isolation procedures 7, 9, 10. However, detailed description of the islet isolation process with visual aid is lacking 7, 9, making it a challenge for most researchers to perform similar studies. 
We have explored several different conditions that yield high quality islets from neonatal mice. Here we present a protocol that is expected to help researchers learn the key details in the islet isolation process. This protocol is applicable to mouse pancreas up to two weeks of age, after which perfusion can be performed for routine islet isolation. Islets can be directly used for insulin secretion and gene expression assays.

<table border="0" cellpadding="0" cellspacing="0" width="769">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:169px;">Collagenase&#160; Type IV</td>
			<td style="width:253px;">Sigma Aldrich, St Louis, MO</td>
			<td style="width:128px;">C5138</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:169px;">1X HBSS with Ca2+/Mg2+</td>
			<td style="width:253px;">Mediatech/Cellgro, Manassas, VA</td>
			<td>MT21020CV</td>
			<td style="width:219px;">If HBSS wihout Ca2+/Mg2+ is obtained, CaCl2 and MgSO4 can be added to 1.26 and 0.5 mM, respectively.&#160;</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:169px;">RPMI 1640 w/o glucose</td>
			<td style="width:253px;">Thermo Fisher Scientific/Life Technologies, Waltham, MA</td>
			<td>11879-020</td>
			<td style="width:219px;">Glucose needs to be added to specific levels to not interfere with seusequent islet usage.&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:169px;">Glucose</td>
			<td style="width:253px;">Sigma Aldrich, St Louis, MO</td>
			<td>G-7021&#160;</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:169px;">polysucrose and sodium diatrizoate solution</td>
			<td style="width:253px;">Sigma Aldrich, St Louis, MO</td>
			<td>Histopaque-10771</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:169px;">Stereoscope</td>
			<td style="width:253px;">Carl-Zeiss, Oberkochen, Germany</td>
			<td>Stemi2000</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:169px;">Stereoscope</td>
			<td style="width:253px;">Leica, Wetzlar, Germany</td>
			<td>Leica M165</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:169px;">Microcentrifuge</td>
			<td style="width:253px;">Eppendorf, Hauppauge, NY</td>
			<td>Centrifuge 5417C</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:169px;">Centrfuge</td>
			<td style="width:253px;">Eppendorf, Hauppauge, NY</td>
			<td>Centrifuge 5810R</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:169px;">15-ml centrfuge tubes&#160;</td>
			<td style="width:253px;">VWR, Radnor, PA</td>
			<td>89039-666</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:169px;">50-ml centrfuge tubes&#160;</td>
			<td style="width:253px;">VWR, Radnor, PA</td>
			<td>89039-658</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:169px;">Precision balance</td>
			<td style="width:253px;">VWR, Radnor, PA</td>
			<td>VWR-225AC</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:169px;">Microfuge tubes</td>
			<td style="width:253px;">VWR, Radnor, PA</td>
			<td>87003-294</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:169px;">Pipetman P1000</td>
			<td style="width:253px;">Fisher Scientific,&#160; Waltham, MA</td>
			<td>F123602</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:169px;">Pipetman P20</td>
			<td style="width:253px;">Fisher Scientific,&#160; Waltham, MA</td>
			<td>F123600</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:169px;">100X15 millimeter dish</td>
			<td style="width:253px;">VWR, Radnor, PA</td>
			<td>25384-088</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:169px;">60X15 millimeter dish</td>
			<td style="width:253px;">VWR, Radnor, PA</td>
			<td>25384-168</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:169px;">12-well plates</td>
			<td style="width:253px;">VWR, Radnor, PA</td>
			<td>665-180</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:169px;">Scissor</td>
			<td style="width:253px;">Fine Scientific Tools, Foster City, CA</td>
			<td>14080-11</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:169px;">Tweezers</td>
			<td style="width:253px;">Fine Scientific Tools, Foster City, CA</td>
			<td>5708-5</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:169px;">CD1 mice</td>
			<td style="width:253px;">Charles River Laboratories, Wilminton, MA&#160;</td>
			<td>CD-1</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:169px;">C57BL/6J</td>
			<td style="width:253px;">The Jackson laboratory, Farmington, CT&#160;</td>
			<td>C57BL/6J</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">B6CBAF1/J</td>
			<td style="width:253px;">The Jackson laboratory, Farmington, CT&#160;</td>
			<td>B6CBAF1/J</td>
			<td style="width:219px;"></td>
		</tr>
	</tbody>
</table>

effective isolation of functional islets from neonatal mouse pancreas

从大型哺乳动物的胰腺基于灌注胰岛隔离协议确立。这种协议是很容易地在许多实验室由于大尺寸的胰管，允许准备注射胶原酶和随后的组织灌注的进行。与此相反，从小型胰腺胰岛分离，像新生小鼠，是具有挑战性，因为灌注是不容易在小胰腺实现的。在这里，我们描述了从恢复视觉协助新生小鼠胰岛相当数量的详细的简单过程。新鲜解剖整个胰腺用在37℃下溶解在Hanks&#39;平衡盐溶液（HBSS）0.5毫克/毫升胶原酶IV消化，在微量离心管。管定期窃听，以帮助组织扩散。当大部分组织的分散，以小簇约1毫米，裂解物，用10％胎牛血清（FBS）洗涤三到四倍用培养基。胰岛集群，缺乏可识别的腺泡组织，然后可以根据解剖立体镜来回收。这种方法恢复20 - 80小到每个刚出生的小鼠胰腺大型小岛。这些胰岛适合大多数可想到下游测定法，包括胰岛素分泌，基因表达，和培养。呈现胰岛素分泌测定法的实例，以验证隔离过程。遗传背景和消化的程度是决定产量的最大因素。高活性新鲜的胶原酶溶液是首选，因为它在内分泌外分泌隔离艾滋病。阳离子在洗涤所有的解决方案和胎牛血清存在下[钙（Ca 2+）和镁（Mg 2+）] /拾取介质所必需的具有适当的完整性胰岛的良好的产率。具有良好的对比度和放大倍率解剖范围也将有所帮助。

从每个小老鼠胰腺胰岛80 - 在最佳条件下，该方法可以产生20。这个数量取决于遗传背景，年龄的小鼠，并加以回收胰岛的大小。这些常用的，CD1出孕育和C57BL / 6J纯种小鼠产生比CD1和C57BL / 6或商业B6CBAF1之间的杂交尺寸更小更少的胰岛/ J小鼠做的。直接用手采摘一般了数量较少的小岛，可能是由于小岛排除这可能是很难当外分泌组织（ 图1A-C）混合的认可。梯?...

Watch this Scientific Journal Video about Effective Isolation of Functional Islets from Neonatal Mouse Pancreas at JoVE.com

Effective Isolation of Functional Islets from Neonatal Mouse Pancreas

我们在此描述了一个协议，从新生小鼠分离胰岛完好无损。胰腺被部分用胶原酶消化，接着洗涤和手采摘。 20 - 80胰岛集群可以每胰腺从新生小鼠，这是适合于几个胰岛研究而获得。

新生小鼠胰腺胰岛功能的有效隔离

Perfusion-based islet-isolation protocols from large mammalian pancreata are well established. Such protocols are readily conducted in many laboratories ...

effective-isolation-of-functional-islets-from-neonatal-mouse-pancreas

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

10.0K 次观看.  Vanderbilt University Medical School. 我们在此描述了一个协议，从新生小鼠分离胰岛完好无损。胰腺被部分用胶原酶消化，接着洗涤和手采摘。 20 - 80胰岛集群可以每胰腺从新生小鼠，这是适合于几个胰岛研究而获得。 

视频: Effective Isolation of Functional Islets from Neonatal Mouse Pancreas

Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is difficult and time consuming. The most commonly used cells are neonatal rat cardiomyocytes (NRCMs), which require isolation every time cells are needed. The birth of the rats can be unpredictable. Cryopreservation is proposed to allow for cells to be stored until needed, yet freezing/thawing methods for primary cardiomyocytes are challenging due to the sensitivity of the cells. Using the proper cryoprotectant, dimethyl sulfoxide (DMSO), cryopreservation was achieved. By slowly extracting the DMSO while thawing the cells, cultures were obtained with viable NRCMs. NRCM phenotype was verified using immunocytochemistry staining for &#945;-sarcomeric actinin. In addition, cells also showed spontaneous contraction after several days in culture. Cell viability after thawing was acceptable at 40-60%. In spite of this, the methods outlined allow one to easily cryopreserve and thaw NRCMs. This gives researchers a greater amount of flexibility in planning experiments as well as reducing the use of animals.

The isolation of neonatal rat cardiomyocytes is a time consuming and unpredictable procedure. This study describes methods for cryopreservation and thawing of neonatal rat cardiomyocytes that allows for more efficient use of cells. The thawed NRCMs can be used for various experiments without the need for performing isolations each time.

隔离和乳鼠心肌细胞冷冻保存

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is ...

科研

发育生物学

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

分离和斑马鱼胚胎单细胞的表征

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

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. ...

Isolation of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

小鼠胚胎Hemogenic内皮细胞的分离

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

Isolation of Type I and Type II Pericytes from Mouse Skeletal Muscles

Pericytes are perivascular multipotent cells that show heterogeneity in different organs or even within the same tissue. In skeletal muscles, there are at least two pericyte subpopulations (called type I and type II), which express different molecular markers and have distinct differentiation capabilities. Using NG2-DsRed and Nestin-GFP double-transgenic mice, type I (NG2-DsRed+Nestin-GFP-) and type II (NG2-DsRed+Nestin-GFP+) pericytes have been successfully isolated. However, the availability of these double-transgenic mice prevents the widespread use of this purification method. This work describes an alternative protocol that allows for the easy and simultaneous isolation of type I and type II pericytes from skeletal muscles. This protocol utilizes the fluorescence-activated cell sorting (FACS) technique and targets PDGFR&#946;, rather than NG2, together with the Nestin-GFP signal. Following isolation, type I and type II pericytes show distinct morphologies. In addition, type I and type II pericytes isolated with this new method, like those isolated from the double-transgenic mice, are adipogenic and myogenic, respectively. These results suggest that this protocol can be used to isolate pericyte subpopulations from skeletal muscles and possibly from other tissues.

This work describes a FACS-based protocol that allows for easy and simultaneous isolation of type I and type II pericytes from skeletal muscles.

从小鼠骨骼肌分离I型和II型周细胞

Pericytes are perivascular multipotent cells that show heterogeneity in different organs or even within the same tissue. In skeletal muscles, there are at ...

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound (30/45MHZ) System

Congenital heart defects (CHDs) are the most common cause of childhood morbidity and early mortality. Prenatal detection&#160;of the underlying molecular mechanisms of CHDs is&#160;crucial for inventing new preventive and therapeutic strategies. Mutant mouse models are powerful tools to discover new&#160;mechanisms&#160;and environmental stress modifiers that drive cardiac development and their potential&#160;alteration in CHDs. However, efforts to establish the causality of these putative contributors have been limited to histological&#160;and molecular studies&#160;in non-survival animal experiments, in which&#160;monitoring the key physiological and hemodynamic parameters is&#160;often absent. Live imaging technology has become an essential tool to establish the&#160;etiology of CHDs. In particular,&#160;ultrasound imaging can be used prenatally without surgically exposing the fetuses,&#160;allowing maintaining&#160;their baseline physiology while monitoring the impact of environmental stress&#160;on the hemodynamic and structural aspects of cardiac chamber development. Herein, we use the High-Frequency Ultrasound (30/45)&#160;system to examine the cardiovascular system in fetal mice at E18.5 in utero at the baseline and in response to prenatal hypoxia exposure. We demonstrate the feasibility of the system to measure cardiac chamber size, morphology, ventricular function, fetal heart rate, and umbilical artery flow indices, and their alterations in fetal mice exposed to systemic chronic hypoxia in utero in real time.

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound 30/45MHZ System

High-frequency ultrasound imaging of the fetal mouse has improved imaging resolution and can provide precise non-invasive characterization of cardiac development and structural defects. The protocol outlined herein is designed to perform real-time fetal mice echocardiography in vivo.

用高频超声 (30/45 mhz) 系统对胎儿小鼠进行心血管成像

Congenital heart defects (CHDs) are the most common cause of childhood morbidity and early mortality. Prenatal detection&#160;of the underlying molecular ...

Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures

Human pluripotent stem cell (hPSC)-derived pancreatic cells are a promising cell source for regenerative medicine and a platform to study human developmental processes. Stepwise directed differentiation that recapitulates developmental processes is one of the major ways to generate pancreatic cells including pancreas/duodenum homeobox protein 1+ (PDX1+) pancreatic progenitor cells. Conventional protocols initiate the differentiation with small colonies shortly after the passage. However, in the state of colonies or aggregates, cells are prone to heterogeneities, which might hamper the differentiation to PDX1+ cells. Here, we present a detailed protocol to differentiate hPSCs into PDX1+ cells. The protocol consists of four steps and initiates the differentiation by seeding dissociated single cells. The induction of SOX17+ definitive endoderm cells was followed by the expression of two primitive gut tube markers, HNF1&#946; and HNF4&#945;, and eventual differentiation into PDX1+ cells. The present protocol provides easy handling and may improve and stabilize the differentiation efficiency of some hPSC lines that were previously found to differentiate inefficiently into endodermal lineages or PDX1+ cells.

Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures

Here, we present a detailed protocol to differentiate human pluripotent stem cells (hPSCs) into pancreas/duodenum homeobox protein 1+ (PDX1+) cells for the generation of pancreatic lineages based on the non-colony type monolayer growth of dissociated single cells. This method is suitable for producing homogenous hPSC-derived cells, genetic manipulation and screening.

高效生成粘附培养中 Hpsc 的胰腺-二叶多布蛋白 1+后后福格内胰腺祖细胞

Human pluripotent stem cell (hPSC)-derived pancreatic cells are a promising cell source for regenerative medicine and a platform to study human ...

Isolation of Specific Neuron Populations from Roundworm Caenorhabditis elegans

During the aging process, many cells accumulate high levels of damage leading to cellular dysfunction, which underlies many geriatric and pathological conditions. Post-mitotic neurons represent a major cell type affected by aging. Although multiple mammalian models of neuronal aging exist, they are challenging and expensive to establish. The roundworm Caenorhabditis elegans is a powerful model to study neuronal aging, as these animals have short lifespan, an available robust genetic toolbox, and well-cataloged nervous system. The method presented herein allows for seamless isolation of specific cells based on the expression of a transgenic green fluorescent protein (GFP). Transgenic animal lines expressing GFP under distinct, cell type-specific promoters are digested to remove the outer cuticle and gently mechanically disrupted to produce slurry containing various cell types. The cells of interest are then separated from non-target cells through fluorescence-activated cell sorting, or by anti-GFP-coupled magnetic beads. The isolated cells can then be cultured for a limited time or immediately used for cell-specific ex vivo analyses such as transcriptional analysis by real time quantitative PCR. Thus, this protocol allows for rapid and robust analysis of cell-specific responses within different neuronal populations in C. elegans.

Isolation of Specific Neuron Populations from Roundworm Caenorhabditis elegans

Here, we present a protocol for simple isolation of specific groups of live neuronal cells expressing green fluorescent protein from transgenic Caenorhabditis elegans lines. This method enables a variety of ex vivo studies focused on specific neurons and has the capacity to isolate cells for further short-term culturing.

特定神经元种群与圆虫卡埃纳哈布迪炎的分离

During the aging process, many cells accumulate high levels of damage leading to cellular dysfunction, which underlies many geriatric and pathological ...

Isolation and Differentiation of Primary Myoblasts from Mouse Skeletal Muscle Explants

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to differentiate into later stages of muscle development. The protocol provided here describes a robust method for the isolation and culture of a highly proliferative population of myoblast cells from young adult mouse skeletal muscle explants. These cells are useful for the study of the metabolic properties of skeletal muscle of different mouse models, as well as in other downstream applications such as transfection with exogenous DNA or transduction with viral expression vectors. The level of differentiation and metabolic profile of these cells depends on the length of exposure, and composition of the media used to induce myoblast differentiation. These methods provide a robust system for the study of mouse muscle cell metabolism ex vivo. Importantly, unlike in vivo models, the methods described here provide a cell population that can be expanded and studied with high levels of reproducibility.

Myoblasts are proliferating precursor cells that differentiate to form polynucleated myotubes and eventually skeletal muscle myofibers. Here, we present a protocol for efficient isolation and culture of primary myoblasts from young adult mouse skeletal muscles. The method enables molecular, genetic, and metabolic studies of muscle cells in culture.

小鼠骨骼肌肉外泄原发性肌基器的分离和分化

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to ...

A Simple and Effective Transplantation Device for Zebrafish Embryos

Classical embryological manipulations, such as removing cells and transplanting cells within or between embryos, are powerful techniques to study complex developmental processes. Zebrafish embryos are ideally suited for these manipulations since they are easily accessible, relatively large in size, and transparent. However, previously developed devices for cell removal and transplantation are cumbersome to use or expensive to purchase. In contrast, the transplantation device presented here is economical, easy to assemble, and simple to use. In this protocol, we first introduce the handling of the transplantation device as well as its assembly from commercially and widely available parts. We then present three applications for its use: generation of ectopic clones to study signal dispersal from localized sources, extirpation of cells to produce size-reduced embryos, and germline transplantation to generate maternal-zygotic mutants. Finally, we show that the tool can also be used for embryological manipulations in other species such as the Japanese rice fish medaka.

Embryological manipulations such as extirpation and transplantation of cells are important tools to study early development. This protocol describes a simple and effective transplantation device to perform these manipulations in zebrafish embryos.

一种简单有效的斑马鱼胚胎移植装置

Classical embryological manipulations, such as removing cells and transplanting cells within or between embryos, are powerful techniques to study complex ...