Name: differentiation of mouse breast epithelial hc11 and eph4 cells
Uploaded: 2020-02-27T12:00:00
Description: Watch this Scientific Journal Video about Differentiation of Mouse Breast Epithelial HC11 and EpH4 Cells at JoVE.com

HC11细胞系由D.Medina博士（德克萨斯州休斯敦）提供。作者感谢女王大学的安德鲁·克雷格博士提供许多试剂和有价值的建议。EpH4细胞是罗斯凯利博士（温哥华UBC）的礼物。科琳·希克为3D文化研究提供了出色的技术援助。
加拿大自然科学和工程研究理事会、加拿大卫生研究院、加拿腺癌基金会（加拿腺癌基金会，安大略省分会）、加拿腺癌研究联盟）的财政援助，安大略省英才中心、乳腺癌行动金斯敦（BCAK）和克莱尔·纳尔逊遗赠基金通过向LR提供赠款，对此表示感谢。BE获得CIHR、CBCF、BCAK和癌症研究协会的资助。PTG由加拿大研究主席、加拿大创新基金会、CIHR、NSERC和加拿大癌症协会提供支持。MN得到了NCIC的特里·福克斯跨学科癌症研究培训项目、研究生奖（QGA）和皇后大学院长奖的支持。MG得到了美国陆军乳腺癌项目、安大略省研究与创新部以及皇后大学咨询研究委员会博士后奖学金的支持。VH得到了CBCF博士奖学金和特里·福克斯基金会跨学科癌症研究培训计划的博士后奖学金的支持，并与CIHR合作。哈纳德·阿丹是NSERC暑期学生班的获得者。BS获得女王大学研究生奖的支持。

1. 电镀 HC11 电池
<ol><li>在使用无菌技术的层流罩中，制备一个装有50 mL HC11细胞培养基的瓶子：RPMI-1640，带10%胎儿牛血清（FBS）、5微克/mL胰岛素和10纳克/mL EGF（见材料表）。</li>
	<li>每3厘米培养皿约400，000个细胞：通过两个10厘米，50%的康料培养皿进入203厘米的菜肴在HC11中等。细胞必须很好地分散，但必须避免干燥。<ol>
<li>使用真空泵将介质吸入烧瓶。</li>
		<li>每10厘米板加入250μL?...

<ol>
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V., Fialka, I., Orci, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+EpH4+mammary+epithelial+cell+subpopulations+which+differ+in+their+morphogenetic+properties.">Isolation of EpH4 mammary epithelial cell subpopulations which differ in their morphogenetic properties.</a> In Vitro Cellular &amp; Developmental Biology - Animal. 34 (6), 468-477 (1998).</li><li>Nagaoka, K., Zhang, H., Watanabe, G., Taya, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+cell+differentiation+regulated+by+MicroRNA-200a+in+mammary+glands.">Epithelial cell differentiation regulated by MicroRNA-200a in mammary glands.</a> PLoS One. 8 (6), 65127 (2013).</li><li>Arulanandam, R., 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=Cadherin-cadherin+engagement+promotes+survival+via+Rac/Cdc42+and+Stat3.">Cadherin-cadherin engagement promotes survival via Rac/Cdc42 and Stat3.</a> Molecular Cancer Research. 17, 1310-1327 (2009).</li><li>Geletu, M., 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=Classical+cadherins+control+survival+through+the+gp130/Stat3+axis.">Classical cadherins control survival through the gp130/Stat3 axis.</a> BBA-Molecular Cell Research. 1833, 1947-1959 (2013).</li><li>Raptis, L., Arulanandam, R., Geletu, M., Turkson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+R(h)oads+to+Stat3:+Stat3+activation+by+the+Rho+GTPases.">The R(h)oads to Stat3: Stat3 activation by the Rho GTPases.</a> Experimental Cell Research. 317 (13), 1787-1795 (2011).</li><li>Raptis, L., 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=Beyond+structure,+to+survival:+Stat3+activation+by+cadherin+engagement.">Beyond structure, to survival: Stat3 activation by cadherin engagement.</a> Biochemistry and Cell Biology. 87, 835-843 (2009).</li><li>Niit, M., 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=Regulation+of+Differentiation+of+HC11+Mouse+Breast+Epithelial+Cells+by+the+Signal+Transducer+and+Activator+of+Transcription-3.">Regulation of Differentiation of HC11 Mouse Breast Epithelial Cells by the Signal Transducer and Activator of Transcription-3.</a> Anticancer Research. 39 (6), 2749-2756 (2019).</li><li>Brinkmann, V., Foroutan, H., Sachs, M., Weidner, K. 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HC11细胞非常适合与肿瘤转化一起进行分化研究。一个额外的优点是HC11细胞很容易感染基于Mo-MLV的抗逆转录病毒载体来表达各种基因。在我们手中，EpH4细胞比HC112更难感染相同的逆转录病毒载体。
细胞与细胞接触和生长抑制是HC11细胞分化的关键先决条件。因此，要在细胞层之间实现均匀的分化，在播种时实现培养皿中细胞的统一分布非常重要。如果需要区?...

在正常组织或肿瘤中，细胞在三维组织中具有大量粘附到邻居的机会，这在培养中被高密度细胞生长所模仿。细胞对细胞粘附主要通过细胞素受体进行介导，后者定义了细胞和组织结构。有趣的是，最近证明，球童在信号转导中也起着强大的作用，特别是在生存信号1中。矛盾的是，最近发现，分化和新发育不良2的细胞对细胞粘附信号中，有一些来自细胞对细胞的粘附信号。在这里，我们描述了两种具有代表性的小鼠乳腺上皮细胞系HC11和EpH4的诱导和评估方法。
HC11小鼠乳腺上皮细胞系可为研究上皮细胞分化提供有用的模型。HC11细胞是一种COMMA-1D衍生的细胞系，起源于中孕巴尔布/c小鼠3的乳腺。与其他COMMA-1D衍生克隆相比，HC11克隆不需要外生添加细胞外基质或与其他细胞类型共增，以便通过乳原激素3体外诱导内源β-酪蛋白基因。这种细胞系在分化研究中被广泛使用，因为它保留了正常乳腺上皮的重要特征：HC11细胞可以在清除的乳腺脂肪垫4中部分重组导管上皮。此外，他们可以区分在二维（2D）培养，当成长为汇合附附在塑料培养皿表面，在类固醇的存在，如Hydrocortisone或德塞马塞松，除了Insulin和Prolactin（HIP介质）缺乏表皮生长因子（EGF），分化5，6，7的抑制剂。在这些条件下，HC11细胞产生β-酪蛋白和WAP等牛奶蛋白，在诱导后4天内通过西方印迹检测。同时，HC11细胞的一部分以随机方式形成基本乳腺状结构，称为"圆顶"。诱导后4~5天可见Domes，并在第10天逐渐增大，同时增加β-酪蛋白产量8。有趣的是，HC11细胞具有突变的p539，因此代表一种前塑性状态。因此，HC11 模型非常适合研究同一细胞系统中的异化信号网络与肿瘤。
EpH4细胞是IM-2细胞的衍生物，是一种非肿瘤细胞系，最初来自自发的细胞乳腺上皮细胞，从中孕的Balb/c小鼠10中分离出来。EpH4细胞在2D培养中形成连续的上皮单层，但不分化成腺状结构10，11。然而，在由EHS小鼠肉瘤细胞12（EHS基质、基质或Matrigel，见材料表）产生的细胞外基质蛋白混合物组成的材料中，经过3D生长后，EpH4细胞还可以重述乳腺分化的初始阶段。在这些条件下，EpH4细胞形成球体（也称为乳腺球体），表现出表基极性和空心流明，能够产生乳蛋白β-酪蛋白和WAP，类似于哺乳期乳腺上皮细胞。与HC11细胞相反，它们未分化，有些表达间质标记13，EpH4细胞表现出纯发光形态14。EpH4细胞也报告通过用地塞米松、胰岛素和泌乳素15的刺激在二维培养中产生牛奶蛋白。然而，这种方法排除了对模仿3D培养中乳腺微环境的调节效应的研究。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>30% Acrylamide/0.8% Bis Solution</td>
			<td>Bio Rad</td>
			<td>1610154</td>
			<td>Western Blotting</td>
		</tr>
		<tr>
			<td>Anti-Cyclin D1 antibody</td>
			<td>rabbit, Santa Cruz</td>
			<td>sc-717</td>
			<td>Western Blotting</td>
		</tr>
		<tr>
			<td>Anti-p120 antibody</td>
			<td>mouse, Santa Cruz</td>
			<td>sc-373751</td>
			<td>Western Blotting</td>
		</tr>
		<tr>
			<td>Anti-&#946; actin antibody</td>
			<td>mouse, Cell Signalling technology</td>
			<td>3700</td>
			<td>Western Blotting</td>
		</tr>
		<tr>
			<td>Anti-&#946; casein antibody</td>
			<td>goat, Santa Cruz Biotechnology</td>
			<td>sc-17971</td>
			<td>Western Blotting</td>
		</tr>
		<tr>
			<td>Aprotinin</td>
			<td>Bio Shop</td>
			<td>APR600</td>
			<td>Lysis Buffer</td>
		</tr>
		<tr>
			<td>Bicinchoninic Acid Solution</td>
			<td>Sigma</td>
			<td>B9643-1L-KC</td>
			<td>Protein Determination</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Bio Shop</td>
			<td>ALB007.500</td>
			<td>Protein Determination</td>
		</tr>
		<tr>
			<td>Clarity Western ECL Substrate</td>
			<td>Bio Rad</td>
			<td>170-5061</td>
			<td>Western Blotting</td>
		</tr>
		<tr>
			<td>Copper(II) sulphate</td>
			<td>Sigma</td>
			<td>C2284-25ML</td>
			<td>Protein Determination</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Thermofisher Scientific</td>
			<td>D1306</td>
			<td>Staining</td>
		</tr>
		<tr>
			<td>Digitonin</td>
			<td>Calbiochem, Cedarlane Laboratories Ltd</td>
			<td>14952-500</td>
			<td>Staining</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Bio Shop</td>
			<td>EDT001.500</td>
			<td></td>
		</tr>
		<tr>
			<td>Epidermal Growth Factor</td>
			<td>Sigma</td>
			<td>E9644</td>
			<td>Medium for HC11 Cells</td>
		</tr>
		<tr>
			<td>Fetal calf serum</td>
			<td>PAA</td>
			<td>A15-751</td>
			<td>Cell Culture Medium</td>
		</tr>
		<tr>
			<td>Goat- Anti Rabbit-HRP</td>
			<td>Santa Cruz</td>
			<td>SC-2004</td>
			<td>Western Blotting</td>
		</tr>
		<tr>
			<td>Hepatocyte Growth Factor recombinant</td>
			<td>Gibco</td>
			<td>PHG0321</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Sigma</td>
			<td>7365-45-9</td>
			<td>Cell Culture</td>
		</tr>
		<tr>
			<td>Horse Anti-Mouse HRP</td>
			<td>Cell Signalling Technology</td>
			<td>7076</td>
			<td>Western Blotting</td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>Sigma</td>
			<td>H0888</td>
			<td>HIP Medium</td>
		</tr>
		<tr>
			<td>insulin</td>
			<td>Sigma</td>
			<td>I6634</td>
			<td>HIP Medium</td>
		</tr>
		<tr>
			<td>Laminar-flow hood</td>
			<td>BioGard Hood</td>
			<td></td>
			<td>Cell Culture</td>
		</tr>
		<tr>
			<td>Leupeptin</td>
			<td>Bio Shop</td>
			<td>LEU001.10</td>
			<td>Lysis Buffer</td>
		</tr>
		<tr>
			<td>Matrix (Engelbreth-Holm-Swarm matrix, Matrigel)</td>
			<td>Corning</td>
			<td>CACB 354230</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Anti-Goat HRP</td>
			<td>Santa Cruz</td>
			<td>sc-8360</td>
			<td>Western Blotting</td>
		</tr>
		<tr>
			<td>Mowiol 4-88 Reagent</td>
			<td>Calbiochem, Cedarlane Laboratories Ltd</td>
			<td>475904-100GM</td>
			<td>Staining</td>
		</tr>
		<tr>
			<td>Multi-photon confocal microscope</td>
			<td>Leica TCS SP2</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Na3VO4</td>
			<td>Bio Shop</td>
			<td>SOV850</td>
			<td>Lysis Buffer</td>
		</tr>
		<tr>
			<td>Na4P207</td>
			<td>Sigma</td>
			<td>125F-0262</td>
			<td>Lysis Buffer</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Bio Shop</td>
			<td>SOD001.1</td>
			<td>Western Blotting</td>
		</tr>
		<tr>
			<td>NaF</td>
			<td>Fisher Scientific</td>
			<td>7681-49-4</td>
			<td>Lysis Buffer</td>
		</tr>
		<tr>
			<td>Nikon digital Camera</td>
			<td>Coolpix 995</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrocellulose</td>
			<td>Bio Rad</td>
			<td>1620112</td>
			<td>Western Blotting</td>
		</tr>
		<tr>
			<td>NP-40</td>
			<td>Sigma</td>
			<td>9016-45-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Fisher Scientific</td>
			<td>30525-89-4</td>
			<td>Staining</td>
		</tr>
		<tr>
			<td>Phase Contrast Microscope</td>
			<td>Olympus IX70</td>
			<td></td>
			<td>Cell Culture</td>
		</tr>
		<tr>
			<td>Phenylmethylsuphonyl fluoride</td>
			<td>Sigma</td>
			<td>329-98-6</td>
			<td>Lysis Buffer</td>
		</tr>
		<tr>
			<td>pMX GFP Rac G12V</td>
			<td>Addgene</td>
			<td>14567</td>
			<td></td>
		</tr>
		<tr>
			<td>Prolactin</td>
			<td>Sigma</td>
			<td>L6520</td>
			<td>HIP Medium</td>
		</tr>
		<tr>
			<td>RPMI-1640</td>
			<td>Sigma</td>
			<td>R8758</td>
			<td>Cell Culture Medium</td>
		</tr>
		<tr>
			<td>Tissue Culture Dish 35</td>
			<td>Sarstedt</td>
			<td>83.3900.</td>
			<td>Cell Culture</td>
		</tr>
		<tr>
			<td>Tissue Culture Plate-24 well</td>
			<td>Sarstedt</td>
			<td>83.1836.300</td>
			<td>Cell Culture</td>
		</tr>
		<tr>
			<td>Transfer Apparatus</td>
			<td>CBS Scientific Co</td>
			<td>EBU-302</td>
			<td>Western Blotting</td>
		</tr>
		<tr>
			<td>Tris Acetate</td>
			<td>Bio Shop</td>
			<td>TRA222.500</td>
			<td>Western Blotting</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sigma</td>
			<td>9002-07-7.</td>
			<td>Cell Culture</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Bio Shop</td>
			<td>TWN510.500</td>
			<td>Western Blotting</td>
		</tr>
		<tr>
			<td>Veritical Gel Electrophoresis System</td>
			<td>CBS Scientific Co</td>
			<td>MGV-202-33</td>
			<td>Western Blotting</td>
		</tr>
	</tbody>
</table>

differentiation of mouse breast epithelial hc11 and eph4 cells

卡塞林在细胞分化和肿瘤的调节中起着重要的作用。在这里，我们描述了两个小鼠乳腺上皮细胞系HC11和EpH4的诱导诱导的起源和方法，以及它们用于研究乳腺发育和肿瘤转化的互补阶段。
HC11小鼠乳腺上皮细胞系源自怀孕的Balb/c小鼠的乳腺。当生长到附着在含有胎儿小牛血清和Hydrocortisone、I nsulin和Prolactin（HIP介质）的介质中附着在塑料培养皿表面时，它与众不同。 在这些条件下，HC11细胞产生乳蛋白β-酪蛋白和乳清酸性蛋白（WAP），类似于哺乳期乳腺上皮细胞，并形成称为"圆顶"的基本乳腺状结构。
EpH4细胞系来自从怀孕的Balb/c小鼠分离出来的自发不朽的小鼠乳腺上皮细胞。与HC11不同，EPH4细胞在HIP培养基的三维（3D）生长条件下培养时，可以完全分化成球体（也称为乳腺球体）。细胞被试模，悬浮在20%的基质中，由恩格尔布雷斯-霍尔姆-斯沃姆（EHS）小鼠肉瘤细胞产生的细胞外基质蛋白混合物组成，镀在一层浓缩基质层之上，涂有塑料培养皿或多孔板，并覆盖一层含有10%基质的HIP培养基。在这些条件下，EpH4细胞形成中空球体，表现出表基极性、空心流明，并产生β-酪蛋白和WAP。
利用这些技术，我们的结果表明，球红/Rac信号的强度对HC11细胞的分化至关重要。虽然 Rac1 是分化所必需的，低水平的激活 RacV12可增加分化，但高 RacV12水平会阻碍分化，同时诱发新奇。相反，EpH4细胞代表了乳腺上皮分化的早期阶段，即使低水平的RacV12也会抑制这种分化。

人们早就知道，上皮细胞和脂肪细胞的分化需要汇合和结合的球形细胞2。我们和其他人证明，细胞对细胞粘附和参与的E-或N-卡塞林和卡塞林-11， 与培养细胞的汇合一样，触发小GTPases Rac和细胞分裂控制蛋白42（Cdc42）的活性急剧增加，这一过程导致白细胞介素+6（IL6）家族细胞因子和Stat3（信号传感器和转录激活器）1，18，19的激活。<s...

Watch this Scientific Journal Video about Differentiation of Mouse Breast Epithelial HC11 and EpH4 Cells at JoVE.com

Differentiation of Mouse Breast Epithelial HC11 and EpH4 Cells

我们描述了两个乳房上皮线HC11和EpH4的分化诱导技术。虽然两者都需要胎儿小牛血清、胰岛素和乳素来产生牛奶蛋白，但EpH4细胞可以在三维培养中完全分化成乳腺球。这些互补模型对于分化和肿瘤的信号转导研究非常有用。

小鼠乳腺皮下HC11和EpH4细胞的分化

Cadherins play an important role in the regulation of cell differentiation as well as neoplasia. Here we describe the origins and methods of the induction ...

HC11和Eep4细胞是小鼠乳房上皮细胞系，可以在培养中分化。同时，这些细胞可以通过许多肿瘤基因进行转化。这些细胞是研究分化与肿瘤转化之间相互关系的理想之选。这些技术可用于信号转导研究，以发现癌症治疗的目标。例如，小GTPase Rac和其他分子可能会触发乳房上皮细胞的转化，当表达到高水平，但令人惊讶的是，在低水平，他们可能会导致分化。其他信号传感器的行为方式可能相似。这些原则可能适用于其他类型的分化以及细胞汇合发挥重要作用，例如脂肪细胞或 myotube 的分化。首次执行此技术的人应记住，电镀 HC11 细胞非常重要。这是因为细胞到细胞的接触对于分化很重要。另一个需要注意的是，从基质中适当提取EepH4细胞对蛋白质定量至关重要，因为任何残留物都会影响蛋白质的测定。此方法的可视化演示很有用，因为协议的一些细节很难在纸上描述。首先根据手稿说明制备一瓶50毫升的HC11细胞培养。要盘板细胞，从五个六厘米50%汇合培养皿中吸出介质，并使用无菌的九英寸棉塞巴氏移液器用约1.8毫升的尝试素清洗细胞。然后每盘添加200微升的三辛，然后旋转板，以分离附着的细胞。观察相位对比显微镜下的细胞与4倍的目标，以确保它们已经开始驱逐。然后用无菌的九英寸巴斯德移液器吸吸约1.5毫升HC11培养基，并在旋转盘中垂直喷对细胞，确保避免溅水。用HC11介质将所有细胞转移到瓶子中，在瓶子中均匀地分散。然后将细胞悬浮液分成20个三厘米的培养皿，每盘移液两毫升细胞。摇动培养皿，将细胞传播开来，并在37摄氏度和5%的二氧化碳中孵育。第二天，当细胞是90-100%的汇合，吸气的介质，并替换为介质与FBS，但没有EGF。在没有ECG的介质中生长细胞24小时后，将分化介质添加到10个菜中，将细胞生长长达10天，保持其他10个菜的对数。每两到三天更换一次，同时使用HIP治疗和控制细胞。要监测分化，请观察具有相位对比度显微镜的细胞。如果细胞表达绿色荧光蛋白，请使用荧光显微镜。此外，通过每天从HIP处理和控制细胞中提取一次蛋白质并进行西式Blot分析，量化分化程度。收集并放置 EpH4 生长介质、EHS 基质和两个锥形 50 毫升管在冰上。在零下20摄氏度下用移液器尖端预培养组织板，并准备EepH4生长介质，在两个50毫升锥形管中补充10或20%基质，并把它们放在冰上，直到准备好使用。在零下20摄氏度下取回预冷组织培养板后，通过用移液器将基质摊开基质，用150微升未稀释的基质覆盖24孔板的10孔。在传播矩阵时避免产生气泡。然后轻轻敲击板的两侧，在37摄氏度下孵育板一小时，使基质凝固。同时，通过尝试性EpH4细胞和前面描述的，确保单细胞悬浮后，再悬浮在三位一体细胞，为分化做好准备。接下来，用血细胞计计算悬浮细胞。将每孔5次10次转移到四个细胞，将1.5毫升无菌锥形离心管转移到1.5千伏无菌圆锥形离心管中，并在250倍g下旋转5分钟。小心吸上一根的介质，将管子放在冰上。使用一毫升移液器尖端重新暂停在350微升的EepH4生长介质的细胞，辅以20%基质，同时保持管子在冰上，确保避免气泡。底层凝固后，将350微升的细胞悬浮液添加到每一个涂层井中，并将其放在37摄氏度的二氧化碳培养箱中一小时，使20%基质层凝固。观察具有相位对比度显微镜的细胞，以确保单个细胞可见。然后加入200微升的EepH4介质，顶部有10%的基质，并在37摄氏度下孵育板。在电镀基质中的细胞一天后开始分化诱导。小心地去除前 10%矩阵介质的 150 微升，添加 200 微升的 EpH4 介质，其中含有 HIP 和 10% 矩阵，对于控件，添加一个 10% 矩阵介质，而不添加 HIP。每两天更换一次介质，用相位对比显微镜监测乳腺层形成，最多10天。为了量化分化，请小心地从井中移掉 10%基体 HIP 介质，然后用 350 微升的冰冷 PBS 冲洗 20%基质层。然后将 70 到 100 微升的冰冷 PBS 与一毫摩尔 EDTA 直接添加到井中，然后用移液器尖端轻轻分离 100% 基质的底部层。在4摄氏度下轻轻摇动盘子30分钟。小心地将球形悬浮液转移到锥形管中，然后用 500 微升 PBS EDTA 冲洗孔，以回收任何剩余的球形。在冰上摇动管子30分钟，确保基质完全溶解。如果看到可见的矩阵团块，添加更多 PBS EDTA 或摇动更长时间。将溶液以 350 倍 g 离心，以对球体进行颗粒状。然后通过西印探吸上一级，对球体进行回旋，探寻细胞中β-卡辛、环素D1和p120RasGAP。在HC11细胞中，对Rac信号的强度存在显著差异依赖性。虽然分化需要内源性 CRAC1，但突变激活 RacV12 的低水平会导致分化能力增加，但高 RacV12 水平会触发分化块并诱发肿瘤。在3D基质培养中探讨EepH4细胞的分化特性时，发现β-casein生产在8至10天达到顶峰，在HIP刺激后4至6天，环素D1表达最大。为了确定单个细胞的定位，它们被染色为DAPI，并用共和显微镜成像。在乳腺层中观察到内细胞死亡和空心流明的形成，而HGF的加入导致管状结构的形成。首次执行此技术的人应记住，HC11 细胞的电镀非常重要。此外，从基质中适当提取 EpH4 细胞对蛋白质定量至关重要，因为任何残留物都会影响蛋白质的测定。此技术可用于研究可能以类似方式运行的其他信号传感器。如果癌症有驱动突变，那么它们完全抑制将没有必要，因为低水平的剩余实际上将诱导分化。

Research

JoVE Journal

Cancer Research

Synthesis and Characterization of an Aspirin-fumarate Prodrug that Inhibits NF&#954;B Activity and Breast Cancer Stem Cells

合成与阿司匹林富马酸酯前药的表征抑制NFκB活动和乳腺癌干细胞

Inflammation is a cancer hallmark that underlies cancer incidence and promotion, and eventually progression to metastasis. Therefore, adding an ...

科研

癌症研究

Invasive Behavior of Human Breast Cancer Cells in Embryonic Zebrafish

在斑马鱼胚胎人乳腺癌细胞的侵袭行为

In many cases, cancer patients do not die of a primary tumor, but rather because of metastasis. Although numerous rodent models are available for studying ...

Live-imaging of Breast Epithelial Cell Migration After the Transient Depletion of TIP60

TIP60 短暂耗竭后乳腺上皮细胞迁移的活体影像学研究

The wound-healing assay is efficient and one of the most economical ways to study cell migration in vitro. Conventionally, images are taken at the ...

Orthotopic Injection of Breast Cancer Cells into the Mice Mammary Fat Pad

乳腺癌细胞原位注射到小鼠乳房脂肪板

A proper animal model is crucial for a better understanding of diseases. Animal models established by different methods (subcutaneous injections, ...

Modeling Breast Cancer via an Intraductal Injection of Cre-expressing Adenovirus into the Mouse Mammary Gland

通过将表达的腺病毒注射到小鼠乳腺腺中,模拟乳腺癌

Breast cancer is a heterogeneous disease, possibly due to complex interactions between different cells of origins and oncogenic events. Mouse models are ...

Evaluating the Differentiation Capacity of Mouse Prostate Epithelial Cells Using Organoid Culture

利用有机体培养评价小鼠前列腺皮细胞的分化能力

The prostate epithelium is comprised predominantly of basal and luminal cells. In vivo lineage tracing has been utilized to define the differentiation ...

Orthotopic Transplantation of Breast Tumors as Preclinical Models for Breast Cancer

乳腺癌临床前模型乳腺肿瘤的矫形移植

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. ...

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

人类乳腺上皮细胞致癌转化的体外评价

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples

从细胞和组织样本中分离和功能评估人类乳腺癌干细胞

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major ...

Studying TGF-&#946; Signaling and TGF-&#946;-induced Epithelial-to-mesenchymal Transition in Breast Cancer and Normal Cells

研究TGF-β信号和TGF-β引起的乳腺癌和正常细胞的上皮到中位过渡

Transforming growth factor-&#946; (TGF-&#946;) is a secreted multifunctional factor that plays a key role in intercellular communication. Perturbations of ...