Name: using primary neurosphere cultures to study primary cilia
Uploaded: 2017-04-14T12:30:00.000+00:00
Duration: 8 min 14 s
Description: Watch this Scientific Journal Video about Using Primary Neurosphere Cultures to Study Primary Cilia at JoVE.com

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?...

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><tbody><tr><td>12 mm round cover glass</td><td>Fisherbrand</td><td>12-545-80&amp;nbsp;</td><td></td></tr><tr><td>24-well plate</td><td>Falcon</td><td>353047</td><td></td></tr><tr><td>4% paraformaldehyde (PFA)</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 &amp;micro;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&#039;s Phosphate-buffered Saline 10X, Modified, without calcium chloride and magnesium chloride, liquid, sterile-filtered, suitable for cell culture</td><td>Sigma</td><td>D1408-500ML</td><td>DPBS</td></tr><tr><td>Dumont #5 Forceps</td><td>Fine science tools</td><td>11254-20</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</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 (200 mM)</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&amp;nbsp;</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 (PLL)</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-100</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>&amp;beta;-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...

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

Universidad San Sebastian

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

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

建立小鼠胚胎神经干细胞培养神经球含量

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

科研

神经科学

One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult neural stem cells in vitro. These assays can be used to compare the precursor potential of cells isolated from genetically different or differentially treated animals&#160;to determine the effects of exogenous factors on neural precursor cell proliferation and differentiation and to generate neural precursor cell lines that can be assayed over continuous passages. The neurosphere assay is traditionally used for the post-hoc identification of stem cells, primarily due to the lack of definitive markers with which they can be isolated from primary tissue&#160;and has the major advantage of giving a quick estimate of precursor cell numbers in brain tissue derived from individual animals. Adherent monolayer cultures, in contrast, are not traditionally used to compare proliferation between individual animals, as each culture is generally initiated from the combined tissue of between 5-8 animals. However, they have the major advantage that, unlike neurospheres, they consist of a mostly homogeneous population of precursor cells and are useful for following the differentiation process in single cells. Here, we describe, in detail, the generation of neurosphere cultures and, for the first time, adherent cultures from individual animals. This has many important implications including paired analysis of proliferation and/or differentiation potential in both the subventricular zone (SVZ) and dentate gyrus (DG) of treated or genetically different mouse lines, as well as a significant reduction in animal usage.

Here we describe a detailed protocol for the simultaneous generation of neural precursor cell cultures, as either adherent monolayers or neurospheres, from the subventricular zone and dentate gyrus of individual adult mice.

一个鼠标，两种文化：隔离和成人神经的培养干细胞从单个小鼠的两个神经源性区

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult ...

2D and 3D Human Induced Pluripotent Stem Cell-Based Models to Dissect Primary Cilium Involvement during Neocortical Development

Primary cilia (PC) are non-motile dynamic microtubule-based organelles that protrude from the surface of most mammalian cells. They emerge from the older centriole during the G1/G0 phase of the cell cycle, while they disassemble as the cells re-enter the cell cycle at the G2/M phase boundary. They function as signal hubs, by detecting and transducing extracellular signals crucial for many cell processes. Similar to most cell types, all neocortical neural stem and progenitor cells (NSPCs) have been shown harboring a PC allowing them to sense and transduce specific signals required for the normal cerebral cortical development. Here, we provide detailed protocols to generate and characterize two-dimensional (2D) and three-dimensional (3D) cell-based models from human induced pluripotent stem cells (hIPSCs) to further dissect the involvement of PC during neocortical development. In particular, we present protocols to study the PC biogenesis and function in 2D neural rosette-derived NSPCs including the transduction of the Sonic Hedgehog (SHH) pathway. To take advantage of the three-dimensional (3D) organization of cerebral organoids, we describe a simple method for 3D imaging of in toto immunostained cerebral organoids. After optical clearing, rapid acquisition of entire organoids allows detection of both centrosomes and PC on neocortical progenitors and neurons of the whole organoid. Finally, we detail the procedure for immunostaining and clearing of thick free-floating organoid sections preserving a significant degree of 3D spatial information and allowing for the high-resolution acquisition required for the detailed qualitative and quantitative analysis of PC biogenesis and function.

We present detailed protocols for the generation and characterization of 2D and 3D human induced pluripotent stem cell (hIPSC)-based models of neocortical development as well as complementary methodologies enabling qualitative and quantitative analysis of primary cilium (PC) biogenesis and function.

基于2D和3D人诱导多能干细胞的模型，用于剖析新皮质发育过程中原发性纤毛受累

Primary cilia (PC) are non-motile dynamic microtubule-based organelles that protrude from the surface of most mammalian cells. They emerge from the older ...

发育生物学

Generation of Neurospheres from Mixed Primary Hippocampal and Cortical Neurons Isolated from E14-E16 Sprague Dawley Rat Embryo

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have a robust in vitro model that can be exploited for various neurobiology studies. Though primary neuron cultures (i.e., long-term hippocampal cultures) have provided scientists with models, it does not yet represent the complexity of brain network completely. In the wake of these limitations, a new model has emerged using neurospheres, which bears a closer resemblance to the brain tissue. The present protocol describes the plating of high and low densities of mixed cortical and hippocampal neurons isolated from the embryo of embryonic day 14-16 Sprague Dawley rats. This allows for the generation of neurospheres and long-term primary neuron culture as two independent platforms to conduct further studies. This process is extremely simple and cost-effective, as it minimizes several steps and reagents previously deemed essential for neuron culture. This is a robust protocol with minimal requirements that can be performed with achievable results and further used for a diversity of studies related to neuroscience.

Presented here is a protocol for the spontaneous generation of neurospheres enriched in neural progenitor cells from high density plated neurons. During the same experiment, when neurons are plated at a lower density, the protocol also results in prolonged primary rat neuron cultures.

从E14-E16斯普拉格·道利大鼠胚胎分离的混合原发希马和皮质神经元生成神经球

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have ...

Isolation and Expansion of Neurospheres from Postnatal (P1&#8722;3) Mouse Neurogenic Niches

The neurosphere assay is an extremely useful in vitro technique for studying the inherent properties of neural stem/progenitor cells (NSPCs) including proliferation, self-renewal and multipotency. In the postnatal and adult brain, NSPCs are mainly present in two neurogenic niches: the subventricular zone (SVZ) lining the lateral ventricles and the subgranular zone of the hippocampal dentate gyrus (DG). The isolation of the neurogenic niches from postnatal brain allows obtaining a higher amount of NSPCs in culture with a consequent advantage of higher yields. The close contact between cells within each neurosphere creates a microenvironment that may resemble neurogenic niches. Here, we describe, in detail, how to generate SVZ- and DG-derived neurosphere cultures from 1&#8722;3-day-old (P1&#8722;3) mice, as well as passaging, for neurosphere expansion. This is an advantageous approach since the neurosphere assay allows a fast generation of NSPC clones (6&#8722;12 days) and contributes to a significant reduction in the number of animal usage. By plating neurospheres in differentiative conditions, we can obtain a pseudomonolayer of cells composed of NSPCs and differentiated cells of different neural lineages (neurons, astrocytes and oligodendrocytes) allowing the study of the actions of intrinsic or extrinsic factors on NSPC proliferation, differentiation, cell survival and neuritogenesis.

In this article, we describe, in detail, a protocol for the generation of neurosphere cultures from postnatal mouse neural stem cells derived from the main mouse neurogenic niches. Neurospheres are used to identify neural stem cells from brain tissue allowing the estimation of precursor cell numbers. Moreover, these 3D structures can be plated in differentiative conditions, giving rise to neurons, oligodendrocytes and astrocytes, allowing the study of cell fate.

产后神经球的分离和扩张 （P1+3） 小鼠神经原性球

The neurosphere assay is an extremely useful in vitro technique for studying the inherent properties of neural stem/progenitor cells (NSPCs) including ...

Efficient and Cost Effective Electroporation Method to Study Primary Cilium-Dependent Signaling Pathways in the Granule Cell Precursor

The primary cilium is a critical signaling organelle found on nearly every cell that transduces Hedgehog (Hh) signaling stimuli from the cell surface. In the granule cell precursor (GCP), the primary cilium acts as a pivotal signaling center that orchestrates precursor cell proliferation by modulating the Hh signaling pathway. The investigation of primary cilium-dependent Hh signaling machinery is facilitated by in vitro genetic manipulation of the pathway components to visualize their dynamic localization to the primary cilium. However, transfection of transgenes in the primary cultures of GCPs using the currently known electroporation methods is generally costly and often results in low cell viability and undesirable transfection efficiency. This paper introduces an efficient, cost-effective, and simple electroporation protocol that demonstrates a high transfection efficiency of ~80-90% and optimal cell viability. This is a simple, reproducible, and efficient genetic modification method that is applicable to the study of the primary cilium-dependent Hedgehog signaling pathway in primary GCP cultures.

Here, we present a reproducible in vitro electroporation protocol for genetic manipulation of primary cerebellar granule cell precursors (GCPs) that is cost-effective, efficient, and viable. Moreover, this protocol also demonstrates a straightforward method for the molecular study of primary cilium-dependent Hedgehog signaling pathways in primary GCP cells.

高效且具有成本效益的电穿孔方法研究颗粒细胞前体中的原代纤毛依赖性信号通路

The primary cilium is a critical signaling organelle found on nearly every cell that transduces Hedgehog (Hh) signaling stimuli from the cell surface. In ...

<meta charset="utf8"></head><body>对从小鼠脑室下区分离的 NSC 进行高效成核

Isolation, Expansion, and Nucleofection of Neural Stem Cells from Adult Murine Subventricular Zone

Isolation and expansion of neural stem cells (NSCs) from the subventricular zone (SVZ) of the adult mouse brain can be achieved in a medium supplemented with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) as mitogens, producing clonal aggregates known as neurospheres. This in vitro system is a valuable tool for studying NSC potential. Transfection of siRNAs or genes carried in plasmids can be used to induce perturbations to gene expression and study NSC biology. However, the exogenous nucleic acid delivery to NSC cultures is challenging due to the low efficiency of central nervous system (CNS) cells transfection. Here, we present an improved nucleofection system that achieves high efficiency of gene delivery in expanded NSCs from adult murine SVZ. We demonstrate that this relatively simple method enhances gene perturbation in adult NSCs, surpassing traditional transfection protocols with survival rates exceeding 80%. Moreover, this method can also be applied in primary isolated NSCs, providing a crucial advancement in gene function studies through gene expression manipulation via knockdown or overexpression in neurosphere cultures.

<meta charset="utf8"></head><body>作者聚焦：改进的核转染技术，在小鼠脑室下区衍生的神经干细胞培养物中实现高效基因递送

Here, we describe a nucleofection system designed to enhance gene delivery efficiency in expanded neural stem cells (NSCs) isolated from the adult murine subventricular zone. The findings demonstrate that this method significantly improves gene perturbation in NSCs, surpassing the effectiveness of traditional transfection protocols and enhancing cell survival rate.

来自成体小鼠脑室下区的神经干细胞的分离、扩增和成核转染

Isolation and expansion of neural stem cells (NSCs) from the subventricular zone (SVZ) of the adult mouse brain can be achieved in a medium supplemented ...

Simple Detection of Primary Cilia by Immunofluorescence

Primary cilia are dynamically regulated during cell cycle progression, specifically during the G0/G1 phases of the cell cycle, being resorbed prior to mitosis. Primary cilia can be visualized with highly sophisticated methods, including transmission electron microscopy, 3D imaging, or using software for the automatic detection of primary cilia. However, immunofluorescent staining of primary cilia is needed to perform these methods. This publication describes a protocol for the easy detection of primary cilia in vitro by staining acetylated alpha tubulin (axoneme) and gamma tubulin (basal body). This immunofluorescent staining protocol is relatively simple and results in high-quality images. The present protocol describes how four cell lines (C2C12, MEF, NHLF, and skin fibroblasts) expressing primary cilia were fixed, immunostained, and imaged with a fluorescent or confocal microscope.

Primary cilia are extracellular structures associated with the centriole. Primary cilia detection by immunofluorescent staining is a relatively simple procedure that results in extremely high-quality images. In this protocol, fibroblasts expressing primary cilia were fixed, immunostained, and imaged in a fluorescent or confocal microscope.

通过免疫荧光简单检测原发性西莉亚

Primary cilia are dynamically regulated during cell cycle progression, specifically during the G0/G1 phases of the cell cycle, being resorbed prior to ...

使用主神经球文化研究纤毛

摘要

探索更多视频

此视频中的章节

建立小鼠胚胎神经干细胞培养神经球含量

一个鼠标，两种文化：隔离和成人神经的培养干细胞从单个小鼠的两个神经源性区

基于2D和3D人诱导多能干细胞的模型，用于剖析新皮质发育过程中原发性纤毛受累

从E14-E16斯普拉格·道利大鼠胚胎分离的混合原发希马和皮质神经元生成神经球

产后神经球的分离和扩张（P1+3）小鼠神经原性球

高效且具有成本效益的电穿孔方法研究颗粒细胞前体中的原代纤毛依赖性信号通路

来自成体小鼠脑室下区的神经干细胞的分离、扩增和成核转染

来自成体小鼠脑室下区的神经干细胞的分离、扩增和成核转染

来自成体小鼠脑室下区的神经干细胞的分离、扩增和成核转染

通过免疫荧光简单检测原发性西莉亚

使用主神经球文化研究纤毛

摘要

探索更多视频

此视频中的章节

建立小鼠胚胎神经干细胞培养神经球含量

一个鼠标，两种文化：隔离和成人神经的培养干细胞从单个小鼠的两个神经源性区

基于2D和3D人诱导多能干细胞的模型，用于剖析新皮质发育过程中原发性纤毛受累

从E14-E16斯普拉格·道利大鼠胚胎分离的混合原发希马和皮质神经元生成神经球

产后神经球的分离和扩张 （P1+3） 小鼠神经原性球

高效且具有成本效益的电穿孔方法研究颗粒细胞前体中的原代纤毛依赖性信号通路

来自成体小鼠脑室下区的神经干细胞的分离、扩增和成核转染

来自成体小鼠脑室下区的神经干细胞的分离、扩增和成核转染

来自成体小鼠脑室下区的神经干细胞的分离、扩增和成核转染

通过免疫荧光简单检测原发性西莉亚

产后神经球的分离和扩张（P1+3）小鼠神经原性球