Work in S.M.'s laboratory is funded by recruitment grants from CPRIT (R1220) and NIH (1R01GM113023-01).

1.从成年小鼠脑中的神经球分离<ol><li>异氟醚过量安乐死的成年小鼠（约2个月）。仔细检查鼠标已停止呼吸而死亡后立即解剖。 </li><li>用剪刀，使正中切口，打开颅骨。取出大脑。 </li><li>放置在冷PBS中脑在冰上的10cm培养皿。按照全安装解剖的方法来获得从侧脑室48 SVZ。 </li><li>放置侧脑室到1.5mL管中，在PBS中添加0.05％胰蛋白酶-EDTA的500μL，并在水浴中温育该管15分钟，在37℃。 </li><li> 15分钟后，添加停止介质的500μL，并轻轻吸管20 - 用1 mL的尖端30倍。避免移液过程中形成气泡。 注意:这一步是细胞生存的关键。 </li><li>降速细胞在500×g下8分钟。弃去上清液，加入1毫升的PBS，重悬吨他的细胞通过用1个毫升尖轻轻吹打5倍。 </li><li>降速在500xg 8分钟。使用1个毫升尖弃去上清液，并添加1毫升基础培养基。 </li><li> （可选）如果细胞碎片中观察到，通过70微米的细胞型过滤通过细胞。 </li><li>计数与血球细胞的数目;一般来说，约30000 - 得到60,000个细胞/ SVZ。 </li><li>板从一个SVZ的细胞进入一个10cm培养皿用10mL NSC培养基和培养物在37℃，5％CO 2。 </li><li> （可选）要避免球体49之间的融合，把1000个细胞在预先填充有1.5毫升NSC培养基和培养物在37℃，5％CO 2的超低结合6孔板的单个孔中。 注:后5-7天，神经球可以观察到（ 图2A）。培养期间可与小鼠的年龄或遗传背景不同。 </li><li>加入2毫升的NSC培养基每3-4天，以保持文化（不要删除现有介质）。 </li></ol> 2.神经球和Ciliogenesis测试的分化能力的分析<ol><li>为了分析分化的能力，分析在分化培养基中附着的条件下，神经球。 </li><li>通过高压灭菌或在使用之前用紫外线照射消毒的12mm圆形盖玻片上。对于贴壁细胞培养物，把一个无菌的12mm圆形盖玻片至无菌条件下在24孔板的孔中。 </li><li>涂层用防护玻璃10秒的0.002％聚-L-赖氨酸（PLL）500μL。吸出溶液，并干燥它10-15分钟。 </li><li>添加的层粘连蛋白溶液（5微克/μL）500μL。孵育1个小时盖玻璃在37℃下。 </li><li>吸出层粘连蛋白和添加分化培养基或NSC培养基（未分化的对照）的500μL。 </li><li>对于分化测定，拿起100 - 与在显微镜下将200μL尖端200微米的球体。加5-10神经球到24孔板中，并培养在分化培养基中7-10天的每个孔中。 </li><li>为了分析未分化的神经球，添加5-10神经球到一个24孔板和文化的每个孔在NSC培养基1-2天。附加神经传播，成长为单层（ 图2B）。 </li><li>小心除去培养基后，固定在PBS中的4％多聚甲醛（PFA）将细胞在室温下15分钟，然后用PBS清洗两次，在室温下5分钟。该板可以被储存在4℃下1-2个月。 注意:为了显现在NSC培养基神经干/祖细胞和分化培养基中分化的细胞，对巢蛋白进行免疫染色（神经干/祖细胞标记物），β微管蛋白III（TUJ1单克隆，神经元标记物），GFAP（胶质纤维酸性蛋白，星形胶质细胞标记物），和O4（少突胶质细胞标记物）（ 图2B-E）。为了分析纤毛，针对Arl13b执行免疫染色（初级CILIA标记）和Gpr161（睫状GPCR）（ 图3）。 </li><li>安装玻璃盖与安装溶液涂布到载玻片。倾斜载玻片以除去过量的溶液。 </li></ol>神经球Ciliogenesis 3.分析<ol><li>为了通过免疫染色分析完整神经球的细胞，转移1mL的培养用培养基的含有多个神经球到1.5mL管和自旋向下在500xg 8分钟。 15分钟除去培养基后固定，用4％（PFA）球体，并用PBS洗涤。降速球体在500×g离心8分钟，收集上清，并在4℃下孵育球体O / N，用30％的蔗糖。 </li><li>使用1个毫升尖弃去上清液，并使用切1mL的前端添加的OCT液500μL。切割尖端的边缘以加宽开口，作为OCT是粘性的。 </li><li>传输含有神经球的OCT溶液到一次性塑料冷冻模具（10毫米×10毫米×5毫米）。 </li><li>冻结m个老上至少15分钟干冰。 </li><li> （可选的）停止实验并在-80℃冷冻机中模具存储长达1年。 </li><li>切用低温恒温器切片;部分的厚度应为15〜30微米。 </li><li>为了可视化在神经球初级纤毛，执行针对Arl13b免疫染色（ 图4）。 </li></ol> 4.文化和神经球和贴壁神经干细胞的通道<ol><li>通道中的神经球，而球体尺寸是100-200微米之间;当神经球过大（300微米或以上），它们不是理想的实验。 </li><li>转移到神经球50mL试管用1个毫升尖端和降速在500xg 8分钟。 </li><li>使用吸气弃去上清液，在PBS中添加0.05％胰蛋白酶-EDTA的500μL，并且在37℃下孵育5分钟。胰蛋白酶的量根据球体的数目而变化。 </li><li>加入血清培养基，轻轻管的500微升吨20次用1个毫升尖。 </li><li>降速在500xg 8分钟。使用1个毫升尖弃去上清液，并添加1毫升基础培养基。 </li><li> （可选）如果细胞碎片或未解离的神经球看出，通过70微米的细胞型过滤通过细胞。 </li><li>通道中的细胞以10,000细胞/ cm 2在NSC培养基中的密度的10cm培养皿;细胞将准备一个星期后的下一个通道。 </li><li> （可选的）要冻结的细胞，添加冷冻介质以产生500,000至1,000,000个细胞/ mL的悬浮液。冷冻用低温冷冻容器中的细胞;未解离的神经也被冻结。 </li><li>对于贴壁培养，dillute细胞以50,000个细胞/ cm 2的上PLL-和层粘连蛋白涂覆有NSC介质盖玻片，并培养1-2天。 </li></ol> 5.饥饿和纤毛的分析<ol><li>制备饥饿培养基（ 表1）。 </li><li>初始PL后1-2天解离后，在一个孔中（对照）和饥饿培养基中再变NSC培养基孔（实验）贴壁细胞的阿婷。 </li><li>文化贴壁细胞24小时。 </li><li>固定在PBS中的4％PFA将细胞在室温下15分钟并用PBS清洗两次，于室温下每次5分钟。 </li><li> （可选的）停止实验并在4℃下用PBS中的盖玻片的24孔板存储用于1-2个月。 </li><li>用于染色7，50执行协议免疫荧光。 </li><li>安装使用安装解决方案的盖玻片。干燥在黑暗载玻片O / N在室温。 </li><li>在必要的放大倍率获取关于化合物显微镜图像。使用显微镜，照相机和目标（40X / 1.3油和63X / 1.4油），使用伴随软件控制。采取足够的z部在0.5-0.8微米的间隔（ 图5）。 </li><li>对于纤毛本地化的定量分析贴壁细胞，通过寻找到DAPI通道获取来自与汇合的细胞连续3-8场中的图像的堆叠。量化使用ImageJ /斐济初级纤毛的数量。典型地，使用在ImageJ的插件"小区计数器"工具&gt;分析对话框来计数与GPCR阳性纤毛细胞;从图像中的堆叠的最大凸起还可以从ImageJ的/ Fiji.Use类似的图像强度和对比度参数导出为来自同一实验的全部图像进行计数和输出的目的。 </li></ol> 6.转染的神经球<ol><li>坚持解离的细胞，以盖玻片24小时。在一个24孔板的单个孔中使用的细胞密度的细胞通常150000之间75000和在500 NSC的μL平台。 </li><li>通过涡旋5秒混合血清培养基减少的25μL和转染试剂的1.5μL在0.5毫升微量离心管。 </li><li>添加2.5微克无内毒素的质粒DNA的一个单独的0.5毫升microtubÈ含有降低血清培养基的25μL和通过涡旋5秒混合。 </li><li>添加转染试剂的1μL到第二含微管DNA和通过涡旋5秒混合。 </li><li>从含有DNA的管到第一微型管中添加混合物中，并通过移液混合。 </li><li>孵育混合物在室温10-15分钟。孵育后，轻轻地转染混合物逐滴添加到孔中，在NSC培养基的顶部（500μL/孔）。 </li><li>改变介质24小时转染后，以控制介质（NSC培养基）或饥饿培养基（500μL/孔）。 </li><li>通过24个小时后更换培养基，以4％PFA固定细胞并进行免疫染色;典型地，至多10％的转染效率使用该方案获得。 </li></ol>

The authors declare no competing financial interests.

在这里，我们描述了一个方法来生成和维护从成年小鼠SVZ神经球文化。是关于文化的几个相关要点如下。首先，球体的尺寸通常50之间 - 200微米。根据我们的经验，当一个神经球得到直径大于300微米，传代的最佳时机已经错过。这些较大的球体包含在核心死细胞。其次，由于神经球通常用于研究神经干/祖细胞，它使用EGF和碱性FGF（bFGF）的，以保持这些细胞的干性是很重要的。因此，因子诱导分化，例如FBS，必须维持神经球的过程中避免。它已经显示，10％FBS可以分化神经球为星形胶质细胞47。三，神经元分化的程度可能取决于鼠标的年龄。如果希望从更多的神经元分化，年轻小鼠（出生后DAÿ0）都可以使用。第四，如果启动新的文化，这是一件好事，检查神经球的分化能力。第五，我们的方法还允许冻融从建立神经球培养为未来的实验使用。解冻后，细胞通常生长为贴壁纺锤状细胞无聚-L-赖氨酸或层粘连蛋白。这可以通过冻融损害细胞引起的。通过前80％汇合 - 重要的是要保持细胞成长，直到他们成为60是很重要的。通过后，细胞通常生长为神经球和一般可以传代高达5 - 10倍。 基于神经球培养方法让我们来研究纤毛贩运和神经干/祖细胞和分化的神经元信号通路。我们在浮动/粘附和差异化的神经检查纤毛。收购几个Z-部分在整个神经的充分程度纤毛可视化是很重要的。此外，船尾ER产生这些神经球的贴壁培养物，和它们挨饿24个小时，大部分细胞是纤毛（ 图5），并在孤儿GPCR，Gpr161富集。然而，长期在亚细胞结构液泡的贴壁培养结果的饥饿。因此，要仔细优化特定的实验目的饥饿时期是非常重要的。 目前，关于ciliogenesis研究中的几个纤毛主要是执行，永生细胞系57。这些细胞系并不总是忠实地概括神经元和神经干/祖细胞表达的信号成分（例如GPCR）或Shh通路机械，使得它必须制定新的方法来研究纤毛的生物过程中的作用。这里描述的基于神经球方法允许我们研究纤毛调节的细胞途径，包括在神经干/祖细胞和DIFF的上下文GPCR和Shh信号erentiated神经元。

初级纤毛是基于微管的动态亚细胞区室，其用作在胚胎神经元发育1,2期间协调细胞信号传导途径，包括音猬因子（Shh）途径感官天线，并且在成年神经元功能3条块亚细胞信号传导，4 。这些信号途径，如修补5的Shh受体的部件;该途径激活平滑（SMO）6;和Gpr161 7，孤儿G蛋白偶联受体（GPCR），所述Shh通路负调节，定位于纤毛以动态方式。多的GPCR据报道，本地化的纤毛神经元在大脑中7，8，9，10 </SUP&gt;，11，12，13，14，15，16。在纤毛和纤毛生成的信号传导途径的缺陷影响多种组织和被统称为ciliopathies 17，18，19。所述ciliopathy疾病谱频繁包括神经发育缺陷，如颅面畸形20，21，22。另外，在丘脑神经元初级纤毛调节饱腹感的中心通路，和缺陷导致中心性肥胖23，在综合征ciliopathies如巴比Biedel综合征24镜像肥胖。此外，神经肽受体纤毛信号调节CENTR人饱腹感通路11，14。如在海马神经元生长抑素受体3腺苷酸环化酶III（ACIII）和GPCR的睫状定位导致新物体识别缺陷和记忆缺陷25,26和平行缺乏睫状完整性27。纤毛生成的信令的发展方面是紧密联系在一起组织动态平衡;特别是，纤毛是在小脑28,29从颗粒祖细胞而产生的Shh亚型髓母细胞瘤的进展重要。因此，纤毛胚胎，产后，和成人的神经元的发育和功能的过程中发挥重要作用。 神经干细胞（NSCs）驻留在脑室下区侧脑室（SVZ），海马的齿状回的颗粒下区，并且在哺乳动物中30，31，32下丘脑第三脑室的脑室区。神经干细胞是多能，具有自我更新的能力，是大脑发育和再生医学30重要。在SVZ最神经干细胞是静态和具有一个孤初级纤毛的是，在许多情况下，伸出到侧脑室33。通过各种受体的定位，诱导下游细胞应答，特别是有关的Shh，TGFβ，和受体酪氨酸激酶途径2，34，35，36中的初级纤毛信号。由于初级纤毛延伸进入侧脑室，假设初级纤毛检测在脑脊液（CSF），以激活神经干细胞的细胞因子37 </s了&gt;。最近的研究表明Shh信号传导途径和初级纤毛是用于干细胞在修复的激活和再生多种组织中，包括嗅上皮，肺，肾和38，39，40，41是至关重要的。然而，通过该CSF的机制与神经干细胞进行通信，并且主是否纤毛参与是未知的。在贴壁培养神经干细胞的纤毛;本地化Shh通路部件，诸如SMO和Gpr161在纤毛;并响应嘘42。因此，神经干细胞可以作为研究Shh通路的重要模型系统，纤毛贩卖和神经元分化的途径。此外，从神经干细胞分化的神经元也可用于睫状贩卖测定。 神经球构成从neura的增殖所产生的自由浮动的细胞群的在特定的生长因子和非粘性表面43，44存在下生长升干/祖细胞。神经球来研究在正常发育和疾病31，45，46，47的神经干/祖细胞用作体外培养模型一样重要。在这里，我们描述了用于培养神经干/祖细胞和分化成神经元/神经胶质基于神经球的测定。我们特别强调信号元件为神经干/祖细胞和分化的神经元（ 图1）的纤毛的贩卖。相对于培养原代神经元，神经球都比较容易培养，是适合于多次传代和冻融循环，并且可以经历分化成神经元/神经胶质。重要的是，我们确定了神经球源性神经干/祖细胞和分化的神经元在纤毛培养和定位信号有关在这些隔室纤毛功能的分子。基于神经球培养方法可作为在神经干细胞和分化的神经元研究ciliogenesis和纤毛贩卖的理想模型系统。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>12 mm round cover glass</td>
			<td>Fisherbrand</td>
			<td>12-545-80&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>24 well plate</td>
			<td>Falcon</td>
			<td>353047</td>
			<td></td>
		</tr>
		<tr>
			<td>4 % paraformaldehyde</td>
			<td>Affymetrix</td>
			<td>19943</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml tube</td>
			<td>Falcon</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr>
			<td>95 mm X 15 mm petri dish, slippable lid</td>
			<td>Fisherbrand</td>
			<td>FB0875714G</td>
			<td>10 cm dish</td>
		</tr>
		<tr>
			<td>70 &#181;m cell strainer</td>
			<td>Falcon</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Affinipure Donkey Anti-Rabbit IgG (H+L)</td>
			<td>Jackson Immunoresearch</td>
			<td>711-545-152</td>
			<td>Donkey anti Rabbit, Alexa 488 secondary antibody</td>
		</tr>
		<tr>
			<td>Arl13B, Clone N295B/66</td>
			<td>Neuromab</td>
			<td>AB_11000053</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Supplement (50X), serum free</td>
			<td>ThermoFisher Scientific</td>
			<td>17504001</td>
			<td>B27</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Thermo scientific</td>
			<td>ST 40R</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryogenic vial</td>
			<td>Corning</td>
			<td>430488</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma</td>
			<td>D9542-10MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I from bovine pancrease</td>
			<td>Sigma</td>
			<td>D5025-15KU</td>
			<td>Dnase I</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Sigma</td>
			<td>D8418-100ML</td>
			<td>DMSO</td>
		</tr>
		<tr>
			<td>Disposable Vinyl Specimen Molds</td>
			<td>Sakura Tissue-Tek Cryomold</td>
			<td>4565</td>
			<td>10 mm X 10 mm X 5 mm</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline 10&#215;, Modified, without calcium chloride and magnesium chloride, liquid, sterile-filtered, suitable for cell culture</td>
			<td>Sigma</td>
			<td>D1408-500ML</td>
			<td>PBS</td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps</td>
			<td>Fine science tools</td>
			<td>11254-20</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS&#160;</td>
			<td>Sigma</td>
			<td>F9026-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoromount-G solution</td>
			<td>Southern Biotech</td>
			<td>0100-01</td>
			<td>mounting solution</td>
		</tr>
		<tr>
			<td>GFAP</td>
			<td>DAKO</td>
			<td>Z0334</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti Mouse IgG1 Secondary Antibody, Alexa Fluor 555 conjugate</td>
			<td>ThermoFisher Scientific</td>
			<td>A-21127</td>
			<td>Goat anti Mouse IgG1, Alexa 555 secondary antibody</td>
		</tr>
		<tr>
			<td>Goat anti Mouse IgG2a Secondary Antibody, Alexa Fluor 555 conjugate</td>
			<td>ThermoFisher Scientific</td>
			<td>A-21137</td>
			<td>Goat anti Mouse IgG2a, Alexa 555 secondary antibody</td>
		</tr>
		<tr>
			<td>Gpr161</td>
			<td>home made</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>human bFGF</td>
			<td>Sigma</td>
			<td>F0291</td>
			<td>FGF</td>
		</tr>
		<tr>
			<td>hemocytometer</td>
			<td>Hausser Scientific</td>
			<td>0.100 mm deep</td>
			<td>improved neubauer</td>
		</tr>
		<tr>
			<td>Isothesia</td>
			<td>Henry Schein</td>
			<td>NDC 11695-0500-2</td>
			<td>Isofluorane</td>
		</tr>
		<tr>
			<td>Laminin from Engelbreth-Holm-Swarm Sarcoma basement membrane</td>
			<td>Sigma</td>
			<td>L2020</td>
			<td>Laminin</td>
		</tr>
		<tr>
			<td>L-Glutamine (200mM)</td>
			<td>Sigma</td>
			<td>G7513</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 3000 Transfection Reagent</td>
			<td>ThermoFisher Scientific</td>
			<td>L3000</td>
			<td></td>
		</tr>
		<tr>
			<td>Mr. Frosty</td>
			<td>Nalgene&#160;</td>
			<td>5100-0036</td>
			<td></td>
		</tr>
		<tr>
			<td>N-2 supplement (100X)</td>
			<td>ThermoFisher Scientific</td>
			<td>17502001</td>
			<td>N2</td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Gibco</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Donkey Serum</td>
			<td>Jackson ImmunoResearch</td>
			<td>017-000-121</td>
			<td></td>
		</tr>
		<tr>
			<td>OCT compound</td>
			<td>Sakura Tissue-Tek</td>
			<td>4583</td>
			<td>OCT</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Sigma</td>
			<td>P4333-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-lysine</td>
			<td>Sigma</td>
			<td>P4707</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human EGF protein, CF</td>
			<td>R and D systems</td>
			<td>236-EG-200</td>
			<td>EGF</td>
		</tr>
		<tr>
			<td>Scissor</td>
			<td>Fine science tools</td>
			<td>14060-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost plus microscope slide</td>
			<td>Fisher scientific</td>
			<td>12-550-15</td>
			<td>slides</td>
		</tr>
		<tr>
			<td>Triton X</td>
			<td>Bio-Rad</td>
			<td>161-0407</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA solution (10X)</td>
			<td>Sigma</td>
			<td>T4174-100</td>
			<td>Trypsin</td>
		</tr>
		<tr>
			<td>COSTAR 6 Well Plate, With Lid Flat Bottom Ultra-Low Attachment Surface Polystyrene, Sterile</td>
			<td>Corning</td>
			<td>3471</td>
			<td>ultra-low binding 6 well plate</td>
		</tr>
		<tr>
			<td>&#946;-tubulin III</td>
			<td>Covance</td>
			<td>MMS-435P</td>
			<td>TUJ1</td>
		</tr>
	</tbody>
</table>

using primary neurosphere cultures to study primary cilia

The primary cilium is fundamentally important for the proliferation of neural stem/progenitor cells and for neuronal differentiation during embryonic, postnatal, and adult life. In addition, most differentiated neurons possess primary cilia that house signaling receptors, such as G-protein-coupled receptors, and signaling molecules, such as adenylyl cyclases. The primary cilium determines the activity of multiple developmental pathways, including the sonic hedgehog pathway during embryonic neuronal development, and also functions in promoting compartmentalized subcellular signaling during adult neuronal function. Unsurprisingly, defects in primary cilium biogenesis and function have been linked to developmental anomalies of the brain, central obesity, and learning and memory deficits. Thus, it is imperative to study primary cilium biogenesis and ciliary trafficking in the context of neural stem/progenitor cells and differentiated neurons. However, culturing methods for primary neurons require considerable expertise and are not amenable to freeze-thaw cycles. In this protocol, we discuss culturing methods for mixed populations of neural stem/progenitor cells using primary neurospheres. The neurosphere-based culturing methods provide the combined benefits of studying primary neural stem/progenitor cells: amenability to multiple passages and freeze-thaw cycles, differentiation potential into neurons/glia, and transfectability. Importantly, we determined that neurosphere-derived neural stem/progenitor cells and differentiated neurons are ciliated in culture and localize signaling molecules relevant to ciliary function in these compartments. Utilizing these cultures, we further describe methods to study ciliogenesis and ciliary trafficking in neural stem/progenitor cells and differentiated neurons. These neurosphere-based methods allow us to study cilia-regulated cellular pathways, including G-protein-coupled receptor and sonic hedgehog signaling, in the context of neural stem/progenitor cells and differentiated neurons.

从在NSC培养基中的SVZ细胞铺板一周后，神经球漂浮观察到（ 图2A）。球体的尺寸50和200微米之间变化。为了检查，如果球体从神经干/祖细胞衍生的，神经球铺板到PLL-和层粘连蛋白包被的在NSC培养基盖玻片2天。然后，他们被免疫染色对神经干/祖细胞标志物，巢。需要有两天，使球体附着于盖玻片并成长为单层细胞。单层细胞为巢蛋白（ 图2B）是阳性的。为了检查神经球的分化能力，我们镀他们以下在部分2中概述到PLL-和步骤层粘连蛋白包被的在分化培养基中的盖玻片没有解离为7-10天。它需要至少一周的分化。神经球附着在盖玻片和扩大编辑成长为单层细胞。我们固定的细胞免疫染色对神经元，星形胶质细胞，少突胶质细胞和标记。我们观察到，分化成β微管蛋白III阳性神经元，GFAP阳性的星形胶质细胞粘附神经球，和O4阳性少突胶质细胞（ 图2C-E）。因此，多能神经球可以从成年小鼠SVZ中导出。 要确定是否粘附神经球和分化的神经元具有初级纤毛，我们免疫染色对Arl13b（初级纤毛标记）51和Gpr161（睫状本地化GPCR）7。根据我们的经验，在表达ARL13B-mCherry的可用的小鼠模型，并与发育中的小鼠大脑皮层的研究中，大多数（如果不是全部）在大脑中的神经元纤毛表达Arl13b，而所有的神经元都没有ACIII阳性的52，53，54。星期四S，我们主要依靠Arl13b检测细胞纤毛。我们检测初级纤毛和Gpr161的在附着神经球（ 图3A）祖细胞的纤毛和在分化的神经元（ 图3B）的定位。因此，粘附的神经球和分化的谱系纤毛在培养物中和定位信号分子。 要确定是否浮动神经球具有初级纤毛，我们冷冻切片漂浮神经球固定后，如在第3部分中概述我们在切片的神经球的神经干/祖细胞（ 图4A-B）观察到的初级纤毛。这是获取整个神经Z-节充分显现纤毛重要，因为它是难以检查，在一个z平面的纤毛。总之，未分化的神经球具有纤毛和非纤毛细胞的异质群体。 图5A-B）中培养。此外，我们在饥饿注意到Gpr161阳性纤毛的数量增加（％Gpr161阳性纤毛/ Arl13b阳性纤毛）（ 图5A-B）。有趣的是，使用未分化的人胚胎干细胞系先前的研究表明，在培养的人胚胎干细胞也具有初级纤毛55。的数量和在这些细胞系在血清饥饿纤毛长度增加，并且这些纤毛也具备的Shh机器的组分。 我们还粘附转染神经干/ progenitoR细胞，如在第6节中概述我们通常发现的高达10％的转染效率，类似于在实验室（ 图6）原代神经元培养物。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 解 </td><td> 零件 </td></tr><tr><td> 1X PBS </td><td>稀释10倍PBS蒸压水</td></tr><tr><td> NSC媒体</td><td> 2毫升50X B27 </td></tr><tr><td></td><td> 1毫升100×N2 </td></tr><tr><td></td><td> 1毫升的200mM L-谷氨酰胺</td></tr><tr><td></td><td> 1毫升100×青霉素/链霉素</td></tr><tr><td></td><td>碱性FGF（人bFGF）（原液25毫微克/毫升在Neurobasal培养基）:20毫微克/毫升或2mg总计100mL的媒体</td></tr><tr><td></td><td> EGF（原液25毫微克/毫升在Neurobasal培养基）:20毫微克/毫升或2mg总计100mL的媒体</td></tr><tr><td></td><td> 95毫升的基础培养基</td></tr><tr><td>人类碱性FGF（人bFGF） </td><td> 1mL至商业人bFGF 25微克瓶基础培养基</td></tr><tr><td></td><td>存储在-20℃或更低的无菌微管。使用单一的80μL等分试样或2微克/ 100mL的NSC介质。 </td></tr><tr><td> EGF </td><td> 8毫升基础培养基添加到商业EGF20μg的瓶</td></tr><tr><td></td><td>存储在-20℃或更低的无菌微管。使用单一的80微克等份/ 100毫升介质。 </td></tr><tr><td>停止媒体</td><td> 1毫升FBS </td></tr><tr><td></td><td> 75ùDNA酶I（75 U /微升在PBS中） </td></tr><tr><td></td><td> 9毫升PBS </td></tr><tr><td>血清培养基</td><td> 1毫升FBS </td></tr><tr><td></td><td> 9 ml基础培养基</td></tr><tr><td>冷冻介质</td><td> 4毫升DMSO </td></tr><tr><td></td><td> 24毫升的基础培养基</td></tr><tr><html><td></td><td> 12毫升30％FBS </td></tr><tr><td>分化培养基</td><td> 90毫升的基础培养基</td></tr><tr><td></td><td> 5毫升5％FBS </td></tr><tr><td></td><td> 1毫升100×青霉素/链霉素</td></tr><tr><td></td><td> 1毫升的200mM L-谷氨酰胺</td></tr><tr><td></td><td> 1毫升100×N2 </td></tr><tr><td></td><td> 2毫升50X B27 </td></tr><tr><td></td><td>碱性FGF（人bFGF）（25纳克的储液/ mL的Neurobasal培养基）:10毫微克/毫升或1毫克总计100毫升</td></tr><tr><td>饥饿培养基</td><td> 95毫升的基础培养基</td></tr><tr><td></td><td> 1毫升100×青霉素/链霉素</td></tr><tr><td></td><td> 1毫升的200mM L-谷氨酰胺</td></tr><tr><td></td><td> 1毫升100×N2 </td></tr><tr><td></td><td> 2毫升50X B27 </td></tr></tbody></table> 表1:解决方案的配方。 ve_content" FO: - together.within页保持= "1"&gt; <img alt="图1" src="/files/ftp_upload/55315/55315fig1.jpg" /> 图1:神经球培养和分化协议的示意图。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55315/55315fig1large.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/55315/55315fig2.jpg" /> 图2:图表神经球分化能力。 （A）代表性神经球被示出。 （B）神经球铺板于在NSC培养基涂覆的层粘连蛋白和PLL-盖玻片2天，并进行免疫染色对巢蛋白（神经干/祖细胞标记物）。 （CE）神经球铺板以下第2节中概述到PLL-和步骤层粘连蛋白包被的在分化培养基中的盖玻片7天无覆盖UT解离;固定;和免疫染色对β微管蛋白III（神经元标记物），GFAP（胶质细胞标记物），和O4（少突胶质细胞标记物）。细胞核被DAPI染色。比例尺=100μm左右。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55315/55315fig2large.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/55315/55315fig3.jpg" /> 图3:神经球和神经元分化的粘附是神经球纤毛。 （A）未离解的神经球铺板于在NSC培养基涂覆的盖玻片2天;固定;免疫染色对Gpr161（绿色），Arl13b（红色），和DNA（蓝色）。 （B）分化的神经元进行免疫染色对Gpr161（绿），Arl13b（红色，A）或β微管蛋白III（红色，B），和DNA（蓝色）。白色箭头指示纤毛Gpr161本地化。在（A）的右图是由白色箭头表示的初级纤毛的放大图。在白色的盒装神经元Gpr161阳性纤毛被示出为在（B）的右侧。标度=10μm的（A）;为50μm（B）。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55315/55315fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/55315/55315fig4.jpg" /> 图4:浮动，未分化的神经球具备纤毛细胞。浮动神经球固定，包埋在OCT，冷冻切片，并进行免疫荧光处理针对Arl13b（红色）和DNA。 在神经球Arl13b阳性初级纤毛被示出。在（A）的面板是14 Z-部在从一个神经球0.8微米的间隔的最大强度投影。在板（B）示出C的放大图iliated细胞在白盒装区域（A）。从所有的Z截面的最大投影图像被示出为堆叠，而数字1至14表示各个Z-部分。白色箭头指示主要纤毛。比例尺=50μm以下。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55315/55315fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/55315/55315fig5.jpg" /> 图5:饥饿诱导Ciliogenesis在解离的贴壁神经干/祖细胞。 （A）分离的细胞接种到在NSC培养基或24个小时饥饿培养基涂覆的盖玻片;固定;免疫染色对Gpr161（绿色），Arl13b（红色），和DNA（蓝色）。 Gpr161阳性纤毛在对照（左）和饥饿（右）细胞中观察到。下部面板处于所述相应的指示的白框的放大图上面板。白色箭头和箭头指示Gpr161阳性和阴性纤毛，分别。比例尺=100μm左右。 Arl13b阳性纤毛细胞（左图）和在Gpr161在控制和饥饿细胞Arl13b阳性纤毛（右图）定位的（B）的定量。既Arl13b阳性纤毛细胞和Gpr161在Arl13b阳性纤毛定位的百分比在饥饿增加。从每个视两个字段从单次实验的两个技术平行进行定量。数据被表示为从视图±标准偏差的所有4场的平均值。 *，P &lt;0.05; **，P &lt;0.01。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55315/55315fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/55315/55315fig6.jpg" /> 图6:在转染解离的贴壁神经干/祖细胞。镀上涂覆的盖玻片上解离的细胞与LAP-TULP3 N末端（1-183个氨基酸）mut12构建体不结合到芯IFT-A复合物56进行转染。介质改为饥饿培养基24小时后转染。将细胞在24个小时后固定，并针对GFP（绿色）进行免疫染色， Gpr161（红），Arl13b（品红），和DNA（蓝色）。与Gpr161-和Arl13b阳性纤毛的转染细胞用白色箭头标记，而没有纤毛的转染细胞被示出具有白箭头。黄色箭头和箭头指向未转染细胞用或不用Gpr161 / Arl13b，分别。比例尺=10μm以下。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55315/55315fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Using Primary Neurosphere Cultures to Study Primary Cilia at JoVE.com

Using Primary Neurosphere Cultures to Study Primary Cilia

初级纤毛是在神经祖细胞增殖，神经细胞的分化，和成人神经元功能至关重要的。这里，我们描述研究ciliogenesis和信号蛋白使用初级神经球培养物在神经干/祖细胞和分化的神经元纤毛的贩卖的方法。

使用主神经球文化研究纤毛

The primary cilium is fundamentally important for the proliferation of neural stem/progenitor cells and for neuronal differentiation during embryonic, ...

using-primary-neurosphere-cultures-to-study-primary-cilia

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

9.3K 次观看.  University of Texas Southwestern Medical Center. 初级纤毛是在神经祖细胞增殖，神经细胞的分化，和成人神经元功能至关重要的。这里，我们描述研究ciliogenesis和信号蛋白使用初级神经球培养物在神经干/祖细胞和分化的神经元纤毛的贩卖的方法。 

视频: Using Primary Neurosphere Cultures to Study Primary Cilia

Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays

Myelination is a complex process that involves both neurons and the myelin forming glial cells, oligodendrocytes in the central nervous system (CNS) and Schwann cells in the peripheral nervous system (PNS). We use an in vitro myelination assay, an established model for studying CNS myelination in vitro. To do this, oligodendrocyte precursor cells (OPCs) are added to the purified primary rodent dorsal root ganglion (DRG) neurons to form myelinating co-cultures. In order to specifically interrogate the roles that particular proteins expressed by oligodendrocytes exert upon myelination we have developed protocols that selectively transduce OPCs using the lentivirus overexpressing wild type, constitutively active or dominant negative proteins before being seeded onto the DRG neurons. This allows us to specifically interrogate the roles of these oligodendroglial proteins in regulating myelination. The protocols can also be applied in the study of other cell types, thus providing an approach that allows selective manipulation of proteins expressed by a desired cell type, such as oligodendrocytes for the targeted study of signaling and compensation mechanisms. In conclusion, combining the in vitro myelination assay with lentiviral infected OPCs provides a strategic tool for the analysis of molecular mechanisms involved in myelination.

Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays

Here we present protocols that offer a flexible and strategic foundation for virally manipulating oligodendrocyte precursor cells to overexpress proteins of interest in order to specifically interrogate their role in oligodendrocytes via the in vitro model of central nervous system myelination.

生产和使用慢病毒的有选择性地转导原少突胶质前体细胞<em&gt;在体外</em&gt;髓鞘测定

Myelination is a complex process that involves both neurons and the myelin forming glial cells, oligodendrocytes in the central nervous system (CNS) and ...

科研

发育生物学

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop within the uterus. A newly developed ex vivo upright droplet culture method provides an attractive alternative to studies performed in utero. The ex vivo droplet culture provides the ability to examine and manipulate cellular interactions and diverse signaling pathways through use of various blocking and activating compounds; additionally, the effects of various pharmacological reagents on the development of specific organs can be studied without unwanted side effects of systemic drug delivery in utero. As compared to other in vitro systems, the droplet culture not only allows for the ability to study three-dimensional morphogenesis and cell-cell interactions, which cannot be reproduced in mammalian cell lines, but also requires significantly less reagents than other ex vivo and in vitro protocols. This paper demonstrates proper mouse fetal organ dissection and upright droplet culture techniques, followed by whole organ immunofluorescence to demonstrate the effectiveness of the method. The ex vivo droplet culture method allows the formation of organ architecture comparable to what is observed in vivo and can be utilized to study otherwise difficult-to-study processes due to embryonic lethality in in vivo models. As a model application system, a small-molecule inhibitor will be utilized to probe the role of vascularization in testicular morphogenesis. This ex vivo droplet culture method is expandable to other fetal organ systems, such as lung and potentially others, although each organ must be extensively studied to determine any organ-specific modifications to the protocol. This organ culture system provides flexibility in experimentation with fetal organs, and results obtained using this technique will help researchers gain insights into fetal development.

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

The&#160;ex vivo&#160;upright droplet culture is an alternative to current&#160;in vitro&#160;and&#160;in vivo&#160;experimental techniques. This protocol is easy to perform and requires smaller amounts of reagent, while permitting the ability to manipulate and study fetal vascularization, morphogenesis, and organogenesis.

运用<em&gt;离体</em&gt;全胎儿各器官的直立滴文化的过程中鼠标研究器官发育过程

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop ...

Generating Primary Fibroblast Cultures from Mouse Ear and Tail Tissues

Primary cells are derived directly from tissue and are thought to be more representative of the physiological state of cells in vivo than established cell lines. However, primary cell cultures usually have a finite life span and need to be frequently re-established. Fibroblasts are an easily accessible source of primary cells. Here, we discuss a simple and quick experimental procedure to establish primary fibroblast cultures from ears and tails of mice. The protocol can be used to establish primary fibroblast cultures from ears stored at RT for up to 10 days. When the protocol is carefully followed, contaminations are unlikely to occur despite the use of non-sterile tissue stored for extended time in some cases. Fibroblasts proliferate rapidly in culture and can be expanded to substantial numbers before undergoing replicative senescence.

We describe a simple and quick experimental procedure for generating primary fibroblasts from the ears and tails of mice. The procedure does not require special animal training and can be used for the generation of fibroblast cultures from ears stored at RT for up to 10 days.

从鼠耳和尾组织生成主成纤维细胞培养

Primary cells are derived directly from tissue and are thought to be more representative of the physiological state of cells in vivo than established cell ...

Human Primary Trophoblast Cell Culture Model to Study the Protective Effects of Melatonin Against Hypoxia/reoxygenation-induced Disruption

This protocol describes how villous cytotrophoblast cells are isolated from placentas at term by successive enzymatic digestions, followed by density centrifugation, media gradient isolation and immunomagnetic purification. As observed in vivo, mononucleated villous cytotrophoblast cells in primary culture differentiate into multinucleated syncytiotrophoblast cells after 72 hr. Compared to normoxia (8% O2), villous cytotrophoblast cells that undergo hypoxia/reoxygenation (0.5% / 8% O2) undergo increased oxidative stress and intrinsic apoptosis, similar to that observed in vivo in pregnancy complications such as preeclampsia, preterm birth, and intrauterine growth restriction. In this context, primary villous trophoblasts cultured under hypoxia/reoxygenation conditions represent a unique experimental system to better understand the mechanisms and signalling pathways that are altered in human placenta and facilitate the search for effective drugs that protect against certain pregnancy disorders. Human villous trophoblasts produce melatonin and express its synthesizing enzymes and receptors. Melatonin has been suggested as a treatment for preeclampsia and intrauterine growth restriction because of its protective antioxidant effects. In the primary villous cytotrophoblast cell model described in this paper, melatonin has no effect on trophoblast cells in normoxic state but restores the redox balance of syncytiotrophoblast cells disrupted by hypoxia/reoxygenation. Thus, human villous trophoblast cells in primary culture are an excellent approach to study the mechanisms behind the protective effects of melatonin on placental function during hypoxia/reoxygenation.

This manuscript presents a unique in vitro model of immunopurified human villous cytotrophoblast cells cultured under hypoxia/reoxygenation. This model is suitable to study the protective effects of promising treatments, such as melatonin, on pregnancy complications associated with increased oxidative stress and altered placental function.

人胎盘滋养层细胞培养模型，研究褪黑素对缺氧的保护作用/复氧引起的中断

This protocol describes how villous cytotrophoblast cells are isolated from placentas at term by successive enzymatic digestions, followed by density ...

Reprogramming Primary Amniotic Fluid and Membrane Cells to Pluripotency in Xeno-free Conditions

Autologous cell-based therapies got a step closer to reality with the introduction of induced pluripotent stem cells. Fetal stem cells, such as amniotic fluid and membrane mesenchymal stem cells, represent a unique type of undifferentiated cells with promise in tissue engineering and for reprogramming into iPSC for future pediatric interventions and stem cell banking. The protocol presented here describes an optimized procedure for extracting and culturing primary amniotic fluid and membrane mesenchymal stem cells and generating episomal induced pluripotent stem cells from these cells in fully chemically defined culture conditions utilizing human recombinant vitronectin and the E8 medium. Characterization of the new lines by applying stringent methods &#8211; flow cytometry, confocal imaging, teratoma formation and transcriptional profiling &#8211; is also described. The newly generated lines express markers of embryonic stem cells &#8211; Oct3/4A, Nanog, Sox2, TRA-1-60, TRA-1-81, SSEA-4 &#8211; while being negative for the SSEA-1 marker. The stem cell lines form teratomas in scid-beige mice in 6-8 weeks and the teratomas contain tissues representative of all three germ layers. Transcriptional profiling of the lines by submitting global expression microarray data to a bioinformatic pluripotency assessment algorithm deemed all lines pluripotent and therefore, this approach is an attractive alternative to animal testing. The new iPSC lines can readily be used in downstream experiments involving the optimization of differentiation and tissue engineering.

This protocol describes the reprogramming of primary amniotic fluid and membrane mesenchymal stem cells into induced pluripotent stem cells using a non-integrating episomal approach in fully chemically defined conditions. Procedures of extraction, culture, reprogramming, and characterization of the resulting induced pluripotent stem cells by stringent methods are detailed.

无异条件下多原代羊水和膜细胞重新编程

Autologous cell-based therapies got a step closer to reality with the introduction of induced pluripotent stem cells. Fetal stem cells, such as amniotic ...

Isolation and Characterization of Primary Rat Valve Interstitial Cells: A New Model to Study Aortic Valve Calcification

Calcific aortic valve disease (CAVD) is characterized by the progressive thickening of the aortic valve leaflets. It is a condition frequently found in the elderly and end-stage renal disease (ESRD) patients, who commonly suffer from hyperphosphatemia and hypercalcemia. At present, there are no medication therapies that can stop its progression. The mechanisms that underlie this pathological process remain unclear. The aortic valve leaflet is composed of a thin layer of valve endothelial cells (VECs) on the outer surfaces of the aortic cusps, with valve interstitial cells (VICs) sandwiched between the VECs. The use of a rat model enables the in vitro study of ectopic calcification based on the in vivo physiopathological serum phosphate (Pi) and calcium (Ca) levels of patients who suffer from hyperphosphatemia and hypercalcemia. The described protocol details the isolation of a pure rat VIC population as shown by the expression of VIC markers: alpha-smooth muscle actin (&#945;-SMA) vimentin and tissue growth factor beta (TGF&#946;) 1 and 2, and the absence of cluster of differentiation (CD) 31, a VEC marker. By expanding these VICs, biochemical, genetic, and imaging studies can be performed to study and unravel the key mediators underpinning CAVD.

This protocol describes the isolation, culture, and calcification of rat-derived valve interstitial cells, a highly physiological in vitro model of calcific aortic valve disease (CAVD). Exploitation of this rat model facilitates CAVD research in exploring the cell and molecular mechanisms that underlie this complex pathological process.

原发性大鼠瓣膜间质细胞的分离与鉴定: 主动脉瓣钙化的新模型研究

Calcific aortic valve disease (CAVD) is characterized by the progressive thickening of the aortic valve leaflets. It is a condition frequently found in ...

Generation and Culturing of Primary Human Keratinocytes from Adult Skin

The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. In addition, keratinocytes play an essential role in the initiation, maintenance, and regulation of epidermal immune responses by being part of the innate immune system responding to antigenic stimuli in a fast, nonspecific manner. Here, we describe a protocol for isolation of primary human keratinocytes from adult skin, and demonstrate that these cells respond to calcium-induced terminal differentiation, as measured by an increased expression of the differentiation marker involucrin. In addition, we show that the isolated keratinocytes are responsive to IL-1&#946;-induced activation of intracellular signaling pathways as measured by the activation of the p38 MAPK pathway. Taken together, we describe a method for isolation and culturing of primary human keratinocytes from adult skin. Because the keratinocytes are the predominant cell type in the epidermis, this method is useful to study molecular mechanisms in cutaneous biology in vitro.

The human skin acts as a first line of defense against the external environment. We present a method for isolating primary human keratinocytes from adult skin. These isolated keratinocytes are useful in numerous experimental setups, and are a highly suitable model for studying molecular mechanisms in cutaneous biology in vitro.

成人皮肤角质形成细胞的生成与培养

The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. ...

Genetic Engineering of Primary Mouse Intestinal Organoids Using Magnetic Nanoparticle Transduction Viral Vectors for Frozen Sectioning

Intestinal organoid cultures provide a unique opportunity to investigate intestinal stem cell and crypt biology in vitro, although efficient approaches to manipulate gene expression in organoids have made limited progress in this arena. While CRISPR/Cas9 technology allows for precise genome editing of cells for organoid generation, this strategy requires extensive selection and screening by sequence analysis, which is both time-consuming and costly. Here, we provide a detailed protocol for efficient viral transduction of intestinal organoids. This approach is rapid and highly efficient, thus decreasing the time and expense inherent in CRISPR/Cas9 technology. We also present a protocol to generate frozen sections from intact organoid cultures for further analysis with immunohistochemical or immunofluorescent staining, which can be used to confirm gene expression or silencing. After successful transduction of viral vectors for gene expression or silencing is achieved, intestinal stem cell and crypt function can be rapidly assessed. Although most organoid studies employ in vitro assays, organoids can also be delivered to mice for in vivo functional analyses. Moreover, our approaches are advantageous for predicting therapeutic responses to drugs because currently available therapies generally function by modulating gene expression or protein function rather than altering the genome.

We describe step-by-step instructions to: 1) efficiently engineer intestinal organoids using magnetic nanoparticles for lenti- or retroviral transduction, and, 2) generate frozen sections from engineered organoids. This approach provides a powerful tool to efficiently alter gene expression in organoids for investigation of downstream effects.

使用磁纳米粒子转导病毒载体进行冷冻切片的原小鼠肠道器官的遗传工程

Intestinal organoid cultures provide a unique opportunity to investigate intestinal stem cell and crypt biology in vitro, although efficient approaches to ...

Induction and Validation of Cellular Senescence in Primary Human Cells

Cellular senescence is a state of permanent cell cycle arrest activated in response to different damaging stimuli. Activation of cellular senescence is a hallmark of various pathophysiological conditions including tumor suppression, tissue remodeling and aging. The inducers of cellular senescence in vivo are still poorly characterized. However, a number of stimuli can be used to promote cellular senescence ex vivo. Among them, most common senescence-inducers are replicative exhaustion, ionizing and non-ionizing radiation, genotoxic drugs, oxidative stress, and demethylating and acetylating agents. Here, we will provide detailed instructions on how to use these stimuli to induce fibroblasts into senescence. This protocol can easily be adapted for different types of primary cells and cell lines, including cancer cells. We also describe different methods for the validation of senescence induction. In particular, we focus on measuring the activity of the lysosomal enzyme Senescence-Associated &#946;-galactosidase (SA-&#946;-gal), the rate of DNA synthesis using 5-ethynyl-2&#39;-deoxyuridine (EdU) incorporation assay, the levels of expression of the cell cycle inhibitors p16 and p21, and the expression and secretion of members of the Senescence-Associated Secretory Phenotype (SASP). Finally, we provide example results and discuss further applications of these protocols.

Here, we discuss a series of protocols for induction and validation of cellular senescence in cultured cells. We focus on different senescence-inducing stimuli and describe the quantification of common senescence-associated markers. We provide technical details using fibroblasts as a model, but the protocols can be adapted to various cellular models.

原发性细胞衰老的诱导与鉴定

Cellular senescence is a state of permanent cell cycle arrest activated in response to different damaging stimuli. Activation of cellular senescence is a ...

A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical applications. The conventional isolation method of human keratinocytes involves a two-step sequential enzymatic digestion procedure, which has been proven to be inefficient in generating primary cells from adult tissues due to the low cell recovery rate and reduced cell viability. We recently reported an advanced method to isolate human primary epidermal progenitor cells from skin tissues that utilizes the Rho kinase inhibitor Y-27632 in the medium. Compared with the traditional protocol, this new method is simpler, easier, and less time-consuming, and increases epithelial stem cell yield and enhances their stem cell characteristics. Moreover, the new methodology does not require the separation of the epidermis from the dermis, and, therefore, is suitable for isolating cells from different types of adult tissues. This new isolation method overcomes the major shortcomings of conventional methods and is more suitable for producing large numbers of epidermal cells with high potency both for laboratory and for clinical applications. Here, we describe the new method in detail.

Here we present a protocol to efficiently isolate primary human keratinocytes from adult skin tissues. This method simplifies the conventional procedure by using the ROCK Inhibitor Y-27632 in the inoculation medium to spontaneously separate epidermal cells from dermal cells.

一种简便有效的成人皮肤组织中人角质形成细胞的分离方法

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical ...