Name: identifying cell surface markers of primary neural stem and progenitor cells by metabolic labeling of sialoglycan
Uploaded: 2019-09-07T12:30:00
Description: Watch this Scientific Journal Video about Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan at JoVE.com

图 1B、1C、1E 和 1F 从白等.10得到英国皇家化学学会的许可。我们感谢X.C.实验室的易浩进行人物编辑。这项工作得到中国国家自然科学基金（第91753206至Q.S.和X.C.，第31371093至Q.S.和21425204和21672013至X.C.）的支持。

本研究中使用的所有动物规程均经清华大学IACUC（机构动物护理和使用委员会）批准，并按照IACUC的准则执行。清华大学实验室动物设施已获得AAALAC（国际实验室动物护理评估与认证协会）的认可。对于胚胎的分期，鉴定的阴道塞的中天被计算为胚胎日0.5（E0.5）。
注：所有细胞在37°C和5%CO2条件下在细胞培养箱中培养。
1. 在渗透支持插?...

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表面标记通常用于标记和纯化体外和体内的特定细胞类型17、18。表面标记物的发现为再生医学和干细胞研究做出了巨大贡献，它提供了分子工具，从正常或病理组织和培养皿中选择性地丰富干细胞群，提供纯化细胞用于临床或研究生物特性的资源。然而，由于难以将干细胞与原发性组织分离，神经干细胞研究的表面标记物开发进展缓慢。此处描述的协?...

神经干细胞被定义为一个多能细胞群，可以自我更新，以维持干细胞库，分化成神经元和胶质。它们是神经系统中的主要细胞类型，通过细胞移植到病变和受伤的大脑1，2，在再生医学中可能提供巨大的治疗潜力。随着发育的进行，神经干细胞群成为异质的3、4，神经干细胞在大脑中的比例逐渐下降5。一般来说，胚胎神经干细胞和其他神经祖细胞，统称为神经干细胞和祖细胞（NSPCs），位于小鼠6的生殖区、心室区和下室区。在胚胎大脑中，神经干细胞通过中间祖细胞（IPC）直接或间接地产生神经元，在某些物种中，通过外部潜区祖体（oRGs）7、8产生神经元。特定的分子特征、形态、干细胞利基的位置和分化潜力都决定了每个亚型在脑器官生成和临床应用中的作用9。然而，目前可用的细胞表面标记不能明确区分和纯化NSPCs的不同亚型，从而限制了对这些亚型的理解和利用。
主要NSPCs表面标记的识别受到三个主要障碍的限制。第一个是组织中NSPCs的细胞数量有限，因此很难制备细胞表面蛋白样本进行常见的质谱分析。第二个限制是难以生成纯细胞亚型，以生成亚型特异性膜蛋白数据。最后，第三个挑战是细胞表面蛋白在全细胞蛋白中的比例低，这妨碍了通过质谱分析的检测灵敏度。
为了克服这些问题，我们开发了一种化学蛋白质组的方法，通过代谢标记的西洛基蛋白质10来选择性地丰富和识别初级NSPC中的细胞表面蛋白。为了产生足够数量的NSPC，我们利用已建立的协议在体外扩大和维护未分化状态的初级胚胎NSPCs，使用可渗透支持与小鼠大脑内皮细胞系共同培养NSPC矩阵插入（例如，透井）系统11。相反，没有内皮细胞单独培养的核动力源产生分化的后代11，12。因此，可以相对分析来自这两个培养系统的蛋白质样本，以识别在NSPCs和分化神经元中不同表达的蛋白质。由于大多数细胞表面蛋白被硅酸13修饰，非天然的硅酸前体模拟N-azido乙酰氨基胺四合化（Ac4ManNAz）被用来劫持内在代谢途径，以便内源性，新的合成的硅石糖被标记为阿齐多组，产生化学手柄14。通过阿齐多-烷基介导的生物正交反应，将生物素与硅脂酶结合，细胞表面蛋白可以通过链球蛋白耦合荧光素或基质14进行可视化和富集，用于蛋白质组鉴定。
在这里，我们执行SDS-PAGE凝胶染色分析表面链胶蛋白酶从NSPC扩展在内皮共培养和区分细胞在非共培养系统中。我们还有选择地在两个培养系统中纯化表面的同步蛋白酶，以便进行蛋白组比较。与传统的离心基细胞表面纯化方案15相比，我们的方案通过特定的标记结合和亲和力减少表面蛋白提取程序，从而提高了提取效率净化。同时，在细胞表面蛋白主要发生细胞表面蛋白的前提下，提高了细胞表面蛋白的提取纯度。虽然内皮因子不能完全阻止扩大的NSPC的分化，但共同培养和分化培养之间的比较研究提供了一种方便的方法来精确定位干细胞富集的表面蛋白，而无需分析由FACS16纯化的NPC蛋白质。我们相信这种方法可以应用于研究其他系统的表面蛋白与适当的修改。

<table border="0" cellpadding="0" cellspacing="0" style="width:880px;" width="879">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">BEND3</td>
			<td style="width:283px;">ATCC</td>
			<td style="width:123px;">CRL-229</td>
			<td style="width:172px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">DMEM</td>
			<td style="width:283px;">Gibco</td>
			<td style="width:123px;">11960044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">L-glutamine</td>
			<td style="width:283px;">Gibco</td>
			<td align="right" style="width:123px;">25030081</td>
			<td align="right">1%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Sodium pyruvate</td>
			<td style="width:283px;">Sigma</td>
			<td style="width:123px;">P5280</td>
			<td align="right">1%</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:303px;">N2 supplement</td>
			<td style="width:283px;">Gibco</td>
			<td align="right" style="width:123px;">17502048</td>
			<td>1 to 100</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:303px;">N-acetyl-L-cysteine</td>
			<td style="width:283px;">Sigma</td>
			<td style="width:123px;">A7250</td>
			<td>1 mM</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:303px;">Papain</td>
			<td style="width:283px;">Worthington</td>
			<td style="width:123px;">LS003726</td>
			<td>10 U/mL</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:303px;">B27 supplement</td>
			<td style="width:283px;">Gibco</td>
			<td align="right" style="width:123px;">17504044</td>
			<td>1 to 50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Poly-L-lysine</td>
			<td style="width:283px;">Sigma</td>
			<td style="width:123px;">P4707</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:303px;">basic Fibroblast growth factor</td>
			<td style="width:283px;">Gibco</td>
			<td style="width:123px;">PHG0261</td>
			<td>10 ng/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Penicillin-Streptomycin</td>
			<td style="width:283px;">Gibco</td>
			<td align="right" style="width:123px;">15140122</td>
			<td align="right">1%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Fetal bovine serum</td>
			<td style="width:283px;">Gibco</td>
			<td style="width:123px;">10099141</td>
			<td align="right">10%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">HBSS</td>
			<td style="width:283px;">Gibco</td>
			<td align="right" style="width:123px;">14175095</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Tripsin-EDTA, 0.25%</td>
			<td style="width:283px;">Gibco</td>
			<td align="right" style="width:123px;">25200056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">DPBS</td>
			<td style="width:283px;">Gibco</td>
			<td align="right" style="width:123px;">14190094</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Transwell</td>
			<td style="width:283px;">Corning</td>
			<td align="right" style="width:123px;">3450</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Paraformaldehyde</td>
			<td style="width:283px;">Sigma</td>
			<td align="right" style="width:123px;">158127</td>
			<td align="right">4%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Sucrose</td>
			<td style="width:283px;">Sangon</td>
			<td style="width:123px;">A100335</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">DAPI</td>
			<td style="width:283px;">Gibco</td>
			<td align="right" style="width:123px;">62248</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">RIPA buffer</td>
			<td style="width:283px;">Thermo Scientific</td>
			<td align="right" style="width:123px;">89900</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">SDS-PAGE loading buffer 2X</td>
			<td style="width:283px;">Solarbio</td>
			<td style="width:123px;">P1018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">6-well plate</td>
			<td style="width:283px;">Corning</td>
			<td align="right" style="width:123px;">3335</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Tris-Glycine protein gel</td>
			<td style="width:283px;">invitrogen</td>
			<td style="width:123px;">xp00100box</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:303px;">mouse monoclonal anti-Nestin</td>
			<td style="width:283px;">Developmental Study Hybridoma Bank</td>
			<td style="width:123px;">Rat-401</td>
			<td style="width:172px;">1 to 20</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:303px;">mouse monoclonal anti-beta-tubulin III</td>
			<td style="width:283px;">Sigma</td>
			<td style="width:123px;">T8860</td>
			<td>1 to 1000</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:303px;">Alexa Fluor 488 goat anti-mouse IgG1</td>
			<td style="width:283px;">invitrogen</td>
			<td style="width:123px;">A-21121</td>
			<td>1 to 1000</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:303px;">Alexa Fluor 546 goat anti-mouse IgG2b</td>
			<td style="width:283px;">invitrogen</td>
			<td style="width:123px;">A-21143</td>
			<td>1 to 1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Albumin Bovine V</td>
			<td style="width:283px;">Amresco</td>
			<td style="width:123px;">0332</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Triton X-100</td>
			<td style="width:283px;">Amresco</td>
			<td style="width:123px;">0694</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">BCA assay kit</td>
			<td style="width:283px;">Thermo Scientific</td>
			<td align="right" style="width:123px;">23225</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">dimethyl sulfoxide</td>
			<td style="width:283px;">Sigma</td>
			<td style="width:123px;">D2650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Brij97</td>
			<td style="width:283px;">Aladdin</td>
			<td style="width:123px;">B129088</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">CuSO4</td>
			<td style="width:283px;">Sigma</td>
			<td align="right" style="width:123px;">209198</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">alkyne-biotin</td>
			<td style="width:283px;">Click Chemistry Tools</td>
			<td style="width:123px;">TA105</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">BTTAA</td>
			<td style="width:283px;">Click Chemistry Tools</td>
			<td align="right" style="width:123px;">1236</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Ac4ManNAz</td>
			<td style="width:283px;">Click Chemistry Tools</td>
			<td align="right" style="width:123px;">1084</td>
			<td>100 &#181;M</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">9AzSia</td>
			<td style="width:283px;">synthesized in lab</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">sodium ascorbate</td>
			<td style="width:283px;">Sigma</td>
			<td style="width:123px;">A4034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Methanol</td>
			<td style="width:283px;">Sigma</td>
			<td align="right" style="width:123px;">34860</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">EDTA</td>
			<td style="width:283px;">Sangon</td>
			<td style="width:123px;">A100322</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">NaCl</td>
			<td style="width:283px;">Sangon</td>
			<td style="width:123px;">A100241</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">SDS</td>
			<td style="width:283px;">Sangon</td>
			<td style="width:123px;">A100227</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Alexa Flour 647-conjugated streptavidin</td>
			<td style="width:283px;">invitrogen</td>
			<td style="width:123px;">S21374</td>
			<td>1 to 1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Triethanolamine</td>
			<td style="width:283px;">Sigma</td>
			<td style="width:123px;">V900257</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Dynabeads M-280 Streptavidin&#160;</td>
			<td style="width:283px;">invitrogen</td>
			<td align="right" style="width:123px;">60210</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">ammonium bicarbonate</td>
			<td style="width:283px;">Sigma</td>
			<td style="width:123px;">9830</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Coomassie Brilliant Blue R-250</td>
			<td style="width:283px;">Thermo Scientific</td>
			<td align="right" style="width:123px;">20278</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Isoflurane</td>
			<td style="width:283px;">RWD Life Science Co.</td>
			<td style="width:123px;">970-00026-00</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:303px;">DNase I</td>
			<td style="width:283px;">Sigma</td>
			<td style="width:123px;">DN25</td>
			<td>12 &#181;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">urea</td>
			<td style="width:283px;">Sigma</td>
			<td style="width:123px;">U5378</td>
			<td></td>
		</tr>
	</tbody>
</table>

identifying cell surface markers of primary neural stem and progenitor cells by metabolic labeling of sialoglycan

神经干细胞和祖细胞（NSPCs）是大脑复杂结构和功能的细胞基础。它们位于体内的专门利基，可以在体外分离和扩展，作为细胞移植修复脑损伤的重要资源。然而，NSPCs是异构的，在分子水平上没有明确定义或由于缺乏特定的细胞表面标记而纯化。先前报道的该协议将神经内皮共培养系统与代谢甘油标记方法相结合，以识别初级NSPCs的表面脂质蛋白酶。NSPC-内皮共培养系统允许在体外自我更新和扩展初级NSPCs，产生足够数量的NSPCs。 培养的NSPC中的Sialoglycan使用非天然的亚西酸代谢报告器进行标记生物正交功能组。通过比较自更新的NSPC中的异二蛋白酶，在内皮共培养中扩展，具有不同的神经培养，我们确定了在NSPCs中丰富的膜蛋白列表。协议详细涉及：1）NSPC内生共文化与NSPC区分文化;2） 标记与阿齐多糖每O-乙酰化N-乙酰氨基苯甲胺（Ac4曼纳兹）;3）生物锡结合到改性双发胶，用于在神经培养固定后进行成像，或从神经培养中提取蛋白质进行质谱分析。然后，通过对扩展NSPC和分化神经培养的质谱数据的比较分析，选取了NSPC富集表面标记候选物。该协议对识别起始材料中低丰度膜蛋白具有高度敏感性，可应用于其他系统具有适当修改的标记发现

原发性胚胎NSPC的体外扩张和代谢标记整个过程需要6天（图1A）。BEND3细胞系和新鲜分离的初级NSPCs的质量是成功实验的关键。BEND3细胞是可溶性因子的来源，可溶性因子刺激NSPCs的自我更新和增殖。在与神经细胞共同培养之前，应确保BEND3细胞没有任何污染，并且以最小的细胞死亡积极分裂。主 NSPC 必须仔细准备，以避免在分离过程中造成过度损坏。?...

Watch this Scientific Journal Video about Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan at JoVE.com

Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan

这里介绍的是一个协议，结合了体外神经内皮共培养系统和代谢结合的异形理念功能组，以扩大主神经干和祖细胞，并标记其表面用于细胞表面标记的成像或质谱分析的紫锥蛋白。

通过Sialoglycan的代谢标记识别主要神经干细胞和祖细胞的细胞表面标记

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized ...

神经干细胞和祖细胞可以自我更新，分化成不同的神经细胞类型，支持神经系统的发展，为再生医学提供资源。这些细胞是异质的，我们需要知道他们特定的细胞表面标记，以获得纯化细胞群并了解其功能。我们的协议提供了一种简单、敏感、高效的技术来分析原发神经干细胞和祖细胞的膜蛋白，帮助识别这些细胞的新表面标记。我们协议的主要优点是能够直接分析原发神经干细胞和祖细胞的表面蛋白质组，提高灵敏度和特异性。这是通过体外内皮细胞中的共培养和高道林内在细胞 MAPK-ERK 通路来标记表面孔隙而实现的。通过孔隙在体外扩张干细胞的亲修饰，我们的协议可以很容易地应用于识别其他干细胞类型的表面标记。为了准备内皮细胞，在90%与9毫升BEN3细胞培养基的汇合下，从10厘米培养皿中重新悬浮BEN3细胞颗粒。将一毫升的细胞悬浮液加入一个可渗透的支撑插件中。在矩阵的底部腔室中，每井再添加两毫升BEN3介质。继续培养细胞在37摄氏度下用5%的二氧化碳培养一天。电镀刀片中的细胞后一天，轻轻吸气介质，首先从底部腔室，然后从刀片。使用预热的 DMEM 清洗刀片的面三次。使用预热的 DMEM 冲洗，清洗刀片的外表面。接下来，将一毫升预热 AM 添加到一个插入中。使用主皮质细胞将刀片转移到孔中。在37摄氏度下用5%的二氧化碳孵育共培养12小时。在 DMSO 中溶解 Ac4ManAz，实现 200 毫摩尔的库存浓度。从培养箱中检索神经内皮共培养板。在每个底腔室中添加一个 Ac4ManAz 库存的微升，每个插入到共培养器中添加 0.5 微升。在37摄氏度下培养细胞，用5%的二氧化碳培养细胞5天。在培养细胞时，每插入物添加100微升RM，每底室添加200微升RM，每隔一天再为内皮细胞和神经细胞重新喂养。首先从共培养板中去除刀片，并从井底吸出培养介质。接下来用预热的PBS清洗神经细胞一次。从油井中吸出 PBS，并在 PBS 中加入一毫升预冷却的 4% 甲状甲醛。在室温下固定细胞10分钟。然后用预冷的PBS清洗细胞三次。吸气PBS，并添加一毫升新鲜准备的生物素结合缓冲液1到每孔。在室温下孵育10分钟。在此之后，从井中吸出反应缓冲液，用PBS洗三次细胞。在细胞的每个井中加入一毫升染色缓冲液，并在室温下孵育30分钟。接下来，从井中吸出染色缓冲液，用预冷却的PBS清洗细胞三次。将一毫升阻断缓冲液加入每个细胞井，并在室温下孵育10分钟。从油井中去除阻塞缓冲液，并在每个油井中加入一毫升的原抗体溶液。在摄氏四度下孵育细胞。第二天，从井中去除原抗体溶液。用预冷的PBS清洗细胞三次。从井中吸出PBS，并在每口井中加入一毫升的二级抗体。在室温下孵育细胞两小时。在此之后，从井中吸出溶液，用预冷的PBS洗三次细胞。首先，准备BTTAA杯硫酸盐复合物2、全蛋白重悬浮缓冲液、蛋白质洗涤缓冲液1、蛋白质洗涤缓冲液2和蛋白质洗涤缓冲液3，如文本协议中所述。接下来，从共培养板中卸下刀片。从底部孔中吸出培养培养，用预冷却的PBS清洗神经细胞一次。吸气 PBS，并在每口井中加入 200 微升预冷却 RIPA 缓冲液。在冰上孵育板5分钟，然后将蛋白质解解收集到1.5毫升管中。在 12000 倍 G 和 4 摄氏度下离心 10 分钟，以对细胞碎片进行颗粒。将上清液转移到新的1.5毫升管中，并根据制造商的说明使用BA试剂盒确定蛋白质浓度。然后，将蛋白质浓度调整为每毫升一毫克。加入100微摩尔氧化铝生物素，2.5毫摩尔钠抗坏血酸和1XBTTAA硫酸盐复合物2至1毫升蛋白质解解。将溶液混合好，在室温下孵育一小时。在此之后，将反应溶液转移到20毫升预冷却甲醇50毫升锥形管中。混合好，在零下30摄氏度过夜孵育，沉淀蛋白质。在4500倍G和4摄氏度下离心15分钟，使蛋白质沉淀。每次洗涤使用 20 毫升预冷却甲醇两次清洗颗粒。接下来，吸上清液，将蛋白质颗粒重新悬浮在四毫升的再悬浮缓冲液中。将蛋白质重新悬浮转移到新的15毫升锥形管中。用PBS洗涤50微升的链球菌珠三次。将洗涤的珠子加入蛋白质重新悬浮，在4摄氏度下以20转/分的旋转速度在垂直旋转器上孵育溶液3小时。在此之后，用此处显示的每个洗涤缓冲液按顺序清洗珠子。用 20 微升 PBS 重新悬浮洗涤的珠子，然后将它们转移到新的 1.5 毫升管中。加入20微升2X蛋白质加载缓冲液，在95摄氏度下治疗10分钟。然后使用SDS-PAGE处理蛋白质样本，然后用文本协议中概述的库马西辉煌蓝染色。在这项研究中，初级胚胎NSPC在体外被扩大和代谢标记。成功的内皮共培养导致 NSPC 形成大型工作表样克隆。抗体的免疫破坏表明，在克隆中，大多数细胞是内辛阳性NSPC，而很少是β-图布林3阳性神经元细胞。相比之下，在非共培养系统中形成的克隆细胞中，内辛阳性细胞和β图布林3个阳性神经元细胞的百分比几乎相同。化学记者Ac4ManAz是一个代谢模拟，可以并入内在蛋白质亚基化通路。使用100微摩尔用于初级NSPC的优化标签浓度，细胞和核形态指示的细胞死亡组合评估表明，这种标记浓度不会引起明显的细胞毒性效应，并且能够有效地标记NSPC。内皮共培养和非共培养系统中的NSPC的克隆形态、自我更新和分化潜力不受影响。从 Ac4ManAz 中通过生物素结合和链球菌珠纯化标记培养物的蛋白质样本在 SDS-PAGE 凝胶中显示了强大的库马西辉煌蓝色染色信号。同时，在装有DMSO对照组的蛋白质样本的车道上，只有染色背景和非特异性结合信号。这也表明 Ac4ManAz 对 NSPC 进行有效标记。在尝试此协议时，生物细胞反应所需的所有溶液都应新鲜准备，反应时间应严格控制，以防止反应过度。按照我们的协议，可以分析神经干细胞富集表面甘油的结构。我们的神经干细胞和祖细胞富集表面蛋白的规程不仅上升标记，而且对凸起模式纯化策略和该细胞群体刚性信号电路的图示也起着重要作用。

Research

JoVE Journal

Neuroscience

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

神经细胞集落形成细胞分析检测，以区分善意的神经祖细胞的神经干细胞

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). ...

科研

神经科学

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

在完整的感觉器官前体细胞的活细胞成像果蝇蛹

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

神经母细胞含量：从区分神经干细胞后代使用流式细胞仪，神经祖细胞的生成和富集含量

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

从小鼠胚胎神经管的神经嵴细胞的分离和培养

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

Assessing Cell Cycle Progression of Neural Stem and Progenitor Cells in the Mouse Developing Brain after Genotoxic Stress

神经干细胞和祖细胞在小鼠脑开发基因毒性应激后的评估细胞周期进程

Neurons of the cerebral cortex are generated during brain development from different types of neural stem and progenitor cells (NSPC), which form a ...

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

从人多能神经嵴前体细胞的无饲养推导干细胞

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

Flow Cytometry Protocols for Surface and Intracellular Antigen Analyses of Neural Cell Types

神经细胞类型的流式细胞仪协议的表面和细胞内抗原分析

Flow cytometry has been extensively used to define cell populations in immunology, hematology and oncology. Here, we provide a detailed description of ...

Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics

神经干细胞和祖细胞来自成年小鼠脑室下区细胞分选和实时成像的细胞周期动力学

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. ...

Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes

人视网膜色素上皮细胞向大鼠眼巩膜视网膜移植精制方案的研制

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is ...

Identifying Transcription Factor Olig2 Genomic Binding Sites in Acutely Purified PDGFR&#945;+ Cells by Low-cell Chromatin Immunoprecipitation Sequencing Analysis

Identifying Transcription Factor Olig2 Genomic Binding Sites in Acutely Purified PDGFRÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â±+ Cells by Low-cell Chromatin Immunoprecipitation Sequencing Analysis

低细胞染色质免疫沉淀测序分析 Olig2 基因组结合位点在急性纯化 PDGFRα + 细胞中的转录因子鉴定

In mammalian cells, gene transcription is regulated in a cell type specific manner by the interactions of transcriptional factors with genomic DNA. ...