Name: isolation of conditionally immortalized mouse glomerular endothelial cells with fluorescent mitochondria
Uploaded: 2022-09-13T13:30:00
Description: Watch this Scientific Journal Video about Isolation of Conditionally Immortalized Mouse Glomerular Endothelial Cells with Fluorescent Mitochondria at JoVE.com

作者感谢何慈江教授和傅佳博士在小鼠内皮细胞分离方面的见解，感谢Mone Zaidi教授为PhAM切除 小鼠提供有价值的讨论。作者还要感谢西奈山伊坎医学院的显微镜CORE和工作人员为我们提供的指导。这项工作得到了美国国立卫生研究院拨款R01DK097253和国防部CDMRP拨款E01 W81XWH2010836对I.S.D.的支持。

这里描述的所有动物程序都得到了西奈山伊坎医学院的IACUC的批准。我们使用从杰克逊实验室购买的三只雄性小鼠（H-2Kb-tsA58转基因小鼠，具有光激活线粒体[PhAM]），并保持正常的食物饮食。
1. 工作条件和准备
<ol><li>使用 70% 乙醇和 UV-C 光清洁层流罩内的工作空间。</li>
	<li>准备珠子的洗涤缓冲液。使用 pH 7.4-8.0 的 0.1 M 磷酸钠制备缓冲液-1，使用 1x 磷酸盐缓冲盐?...

<ol>
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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+in+early+diabetic+kidney+disease:+Glomerular+endothelial+cell+dysfunction.">Molecular mechanisms in early diabetic kidney disease: Glomerular endothelial cell dysfunction.</a> International Journal of Molecular Sciences. 21 (24), 9456 (2020).</li><li>Haraldsson, B., Jeansson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glomerular+filtration+barrier.">Glomerular filtration barrier.</a> Current Opinion in Nephrology and Hypertension. 18 (4), 331-335 (2009).</li><li>Yilmaz, O., Afsar, B., Ortiz, A., Kanbay, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+endothelial+glycocalyx+in+health+and+disease.">The role of endothelial glycocalyx in health and disease.</a> Clinical Kidney Journal. 12 (5), 611-619 (2019).</li><li>Giacco, F., Brownlee, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress+and+diabetic+complications.">Oxidative stress and diabetic complications.</a> Circulation Research. 107 (9), 1058-1070 (2010).</li><li>Daehn, I. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glomerular+endothelial+cell+stress+and+cross-talk+With+podocytes+in+early+[corrected]+diabetic+kidney+disease.">Glomerular endothelial cell stress and cross-talk With podocytes in early [corrected] diabetic kidney disease.</a> Frontiers in Medicine. 5, 76 (2018).</li><li>Satchell, S. 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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=Isolation+and+characterization+of+conditionally+immortalized+mouse+glomerular+endothelial+cell+lines.">Isolation and characterization of conditionally immortalized mouse glomerular endothelial cell lines.</a> Kidney International. 66 (6), 2193-2201 (2004).</li><li>Pham, A. H., McCaffery, J. M., Chan, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+lines+with+photo-activatable+mitochondria+to+study+mitochondrial+dynamics.">Mouse lines with photo-activatable mitochondria to study mitochondrial dynamics.</a> Genesis. 50 (11), 833-843 (2012).</li><li>Archer, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+dynamics--Mitochondrial+fission+and+fusion+in+human+diseases.">Mitochondrial dynamics--Mitochondrial fission and fusion in human diseases.</a> The New England Journal of Medicine. 369 (23), 2236-2251 (2013).</li><li>Casalena, G. A., 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=The+diabetic+microenvironment+causes+mitochondrial+oxidative+stress+in+glomerular+endothelial+cells+and+pathological+crosstalk+with+podocytes.">The diabetic microenvironment causes mitochondrial oxidative stress in glomerular endothelial cells and pathological crosstalk with podocytes.</a> Cell Communication and Signaling. 18 (1), 105 (2020).</li><li>Qi, H., 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=Glomerular+endothelial+mitochondrial+dysfunction+is+essential+and+characteristic+of+diabetic+kidney+disease+susceptibility.">Glomerular endothelial mitochondrial dysfunction is essential and characteristic of diabetic kidney disease susceptibility.</a> Diabetes. 66 (3), 763-778 (2017).</li><li>Akis, N., Madaio, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+culture,+and+characterization+of+endothelial+cells+from+mouse+glomeruli.">Isolation, culture, and characterization of endothelial cells from mouse glomeruli.</a> Kidney International. 65 (6), 2223-2227 (2004).</li><li>Schuler, M. 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线粒体对细胞代谢、稳态和应激反应至关重要，其功能障碍与许多疾病有关，包括肾脏疾病。线粒体在过量活性氧（ROS）的病理生成，细胞内钙水平，细胞死亡途径和细胞骨架动力学的调节中起作用21，22，23。
小鼠GEC的分离具有挑战性，因为它们的数量，大小，基质因子很少，并且与其他肾小球细胞的相?...

肾小球通过限制大分子通过肾小球滤过屏障而对血液滤过至关重要1，2。肾小球包含四种细胞类型：壁上皮细胞、足细胞（内脏上皮细胞）、肾小球内皮细胞 （GEC） 和系膜细胞3。肾小球内皮的特征在于独特的血管结构，根据大过滤量所需的Fenestrae的存在4。肾小球内皮的顶端表面覆盖着带负电荷的糖萼层和称为内皮表层的涂层，该涂层在内皮和血液之间形成空间。这种结构提供了高电荷选择性，限制了带负电荷的分子（如白蛋白）的通过，并防止了白细胞和血小板粘附5。
GEC对代谢变化非常敏感，例如与糖尿病环境相关的高血糖症。事实上，糖尿病会导致有害物质循环增加，葡萄糖代谢途径饱和，并扰乱细胞氧化还原平衡3，6。此外，活性氧的增加诱发线粒体功能障碍，从而影响内皮功能7。
当前方案的总体目标是分离具有荧光线粒体特征的永生化肾小球内皮细胞。事实上，原代GEC的细胞培养具有有限的增殖周期和早期衰老8。此外，荧光线粒体的存在有助于检查高血糖或任何其他治疗的裂变和融合事件。作为一种替代方法，其他实验室使用h-TERT在体外使细胞永生化9。
这里描述的方法允许从4-6周龄的动物中分离有条件永生化的mitoDendra2肾小球内皮细胞（图1）。该详细协议描述了使用携带猿病毒40大肿瘤抗原（SV40 TAg）基因10，11的转基因小鼠（H-2K b-tsA58）来产生热不稳定的条件永生化细胞。tsA58 TAg基因产物在小鼠H-2Kb基因的诱导5'侧翼启动子的控制下在33°C的允许温度下起作用，该启动子在暴露于干扰素γ（IFNγ）时升高到基础水平以上，因此维持条件增殖表型12。在没有IFNγ的情况下，H-2Kb在37°C的非允许温度下迅速降解，消除了细胞中tsA58Tag的永生化功能，并允许细胞发展出更分化的表型13，14，15。H-2Kb-tsA58转基因小鼠与PhAM小鼠的选择性杂交，PhAM小鼠表达线粒体特异性（细胞色素c氧化酶亚基VIII）Dendra2-green，允许活检测荧光线粒体16。Dendra2 绿色荧光在暴露于 405 nm 激光16 后切换到红色荧光。当线粒体在光开关后融合时，它们形成细长的形状，由于绿色和黄色物质的交换而呈现黄色，或者在发生裂变时呈现红色7，17。mitoDendra2-GEC是研究GEC线粒体对不同刺激的细胞反应的好工具。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100 &#181;m cell strainer</td>
			<td>Fisher</td>
			<td>22-363-549</td>
			<td></td>
		</tr>
		<tr>
			<td>1ml Insulin Syringes</td>
			<td>BD</td>
			<td>329424</td>
			<td></td>
		</tr>
		<tr>
			<td>25G butterfly</td>
			<td>BD</td>
			<td>367298</td>
			<td></td>
		</tr>
		<tr>
			<td>3 mm cutting edge scissors</td>
			<td>F.S.T</td>
			<td>15000-00</td>
			<td></td>
		</tr>
		<tr>
			<td>30ml syringe</td>
			<td>BD Biooscience</td>
			<td>309650</td>
			<td></td>
		</tr>
		<tr>
			<td>40 &#181;m cell strainer</td>
			<td>Fisher</td>
			<td>22-363-547</td>
			<td></td>
		</tr>
		<tr>
			<td>40 &#181;m nylon mesh</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bonn Scissors</td>
			<td>F.S.T</td>
			<td>14184-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Fisher</td>
			<td>BP1600-100</td>
			<td></td>
		</tr>
		<tr>
			<td>CD31</td>
			<td>abcam</td>
			<td>ab7388</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase type I</td>
			<td>Corning</td>
			<td>354236</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase type II</td>
			<td>SIGMA</td>
			<td>C6885</td>
			<td>125CDU/mg</td>
		</tr>
		<tr>
			<td>Collagene type IV</td>
			<td>SIGMA</td>
			<td>C5533-5M</td>
			<td></td>
		</tr>
		<tr>
			<td>Dnase-I</td>
			<td>Qiagen</td>
			<td>79254</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynabeads 450</td>
			<td>Thermofisher Scientific</td>
			<td>14013</td>
			<td></td>
		</tr>
		<tr>
			<td>endothelial cells growth medium</td>
			<td>Lonza</td>
			<td>cc-3156</td>
			<td></td>
		</tr>
		<tr>
			<td>Extra fine graefe forceps</td>
			<td>F.S.T</td>
			<td>11150-10</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gemini</td>
			<td>100-106</td>
			<td>Heat inactivated</td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Thermofisher</td>
			<td>33016015</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>F.S.T Dumont</td>
			<td>E6511</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>GIBCO</td>
			<td>14065-056</td>
			<td></td>
		</tr>
		<tr>
			<td>IFNg</td>
			<td>Cell Science</td>
			<td>CRI001B</td>
			<td></td>
		</tr>
		<tr>
			<td>Immortomouse</td>
			<td>Jackson laboratory</td>
			<td>32619</td>
			<td>Tg(H2-K1-tsA58)6Kio/LicrmJ</td>
		</tr>
		<tr>
			<td>L-Glutamine 100x</td>
			<td>Thermofisher Scientific</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic particle concentrator</td>
			<td>Thermofisher Scientific</td>
			<td>12320D</td>
			<td></td>
		</tr>
		<tr>
			<td>mitotracker</td>
			<td>Thermofisher Scientific</td>
			<td>M7512</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS 1X</td>
			<td>Corning</td>
			<td>46-013-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>penecillin streptomycin 100x</td>
			<td>Thermofisher Scientific</td>
			<td>10378016</td>
			<td></td>
		</tr>
		<tr>
			<td>PhaM mice</td>
			<td>Jackson laboratory</td>
			<td>18397</td>
			<td>B6;129S-Gt(ROSA)26Sortm1.1(CAG-COX8A/Dendra2)Dcc/J</td>
		</tr>
		<tr>
			<td>Protease (10 mg/ml)</td>
			<td>SIGMA</td>
			<td>P6911</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>GIBCO</td>
			<td>3945</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyruvate 100mM</td>
			<td>Thermofisher Scientific</td>
			<td>11360070</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard pattern forceps</td>
			<td>&#160;F.S.T</td>
			<td>11000-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Scissors - Sharp-Blunt</td>
			<td>F.S.T</td>
			<td>14008-14</td>
			<td></td>
		</tr>
		<tr>
			<td>synaptopodin</td>
			<td>Santa Cruz</td>
			<td>sc-515842</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin 0.05%</td>
			<td>Thermofisher Scientific</td>
			<td>25300054</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of conditionally immortalized mouse glomerular endothelial cells with fluorescent mitochondria

肾小球内皮细胞（GEC）功能障碍可引发并导致肾小球滤过屏障破坏。线粒体氧化应激增加被认为是导致某些肾小球疾病发病机制中GEC功能障碍的机制。从历史上看，由于难以从肾小球中分离纯培养物，因此从 体内 模型中分离GECs一直具有挑战性。GEC在 体外 具有复杂的生长要求，并且寿命非常有限。在这里，我们描述了用荧光线粒体分离和培养有条件永生化GEC的程序，从而能够跟踪线粒体裂变和融合事件。GECs从表达不稳定性的SV40 TAg（来自Immortomouse）的双转基因小鼠的肾脏中分离，有条件地促进增殖和抑制细胞分化，以及所有线粒体中的光转换荧光蛋白（Dendra2）（来自光激活线粒体[PhAM切除]小鼠）。产生的稳定细胞系允许在永生化SV40 TAg基因失活和线粒体亚群的光活化后进行细胞分化，从而导致荧光从绿色切换到红色。使用mitoDendra2-GEC可以对荧光线粒体的分布，融合和裂变事件进行实时成像，而不会对细胞进行染色。

在本文中，描述了用于分离具有稳定荧光线粒体（mitoDendra2-GEC）的条件永生化肾小球内皮细胞的详细方案（图1）。使用年轻的6-10周龄小鼠对于获得大量健康细胞至关重要。培养3天后，细胞开始从分离的肾小球缓慢生长，如图3G所示。7天后，细胞呈异质性，显示其他肾小球细胞类型，如足细胞，壁上皮细胞和系膜细胞（图4A）。?...

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Isolation of Conditionally Immortalized Mouse Glomerular Endothelial Cells with Fluorescent Mitochondria

本文描述了从表达热不稳定猿病毒40和光激活线粒体PhAM切除的转基因小鼠的肾脏中分离条件永生化肾小球内皮细胞的方法。我们描述了使用磁珠、消化步骤、播种和培养 GEC-CD31 阳性从整个肾脏中分离肾小球的程序。

用荧光线粒体分离条件永生化的小鼠肾小球内皮细胞

Glomerular endothelial cell (GEC) dysfunction can initiate and contribute to glomerular filtration barrier breakdown. Increased mitochondrial oxidative ...

具有稳定荧光线粒体的永生化肾小球内皮细胞是体外检查不同刺激对线粒体结构影响的好工具。本文中描述的方法产生大量的肾小球内皮细胞。通过在GEC上测试小分子，我们可以筛查GEC受到影响的糖尿病肾病的单一疗法。所描述的方法易于执行，不需要特定的设备。但是，重要的是要遵循所述步骤并对设备进行消毒以避免污染。首先，将分离肾小球细胞所需的灌注和分离材料放置在工作空间上。用0.2微米的过滤器过滤新鲜制备的汉克斯平衡盐溶液（HBSS），并将其放在引擎盖下以避免污染。然后，将200微升磁珠与20毫升HBSS混合。用10%FCS和内皮细胞培养基在37摄氏度的水浴中预热RPMI。然后，在室温下用两毫升每毫升10微克胶原蛋白IV预涂六孔板的两个孔45分钟。用PBS清洗盘子两次，以去除用于稀释胶原蛋白的乙酸。立即使用涂层板，或将其在 2 到 8 摄氏度下存放长达四周。用70%乙醇清洁手术器械和工作空间。随后，将手术器械高压灭菌30分钟。打开麻醉小鼠的腹部后，将蝶针插入左心室。要注射100微升珠溶液以扩大血液循环，请小心地用19号针头打破肾脏下方的下腔静脉，并继续灌注珠溶液。接下来，用镊子轻轻去除脂肪组织，用细手术剪刀C切除两个肾脏，然后将它们放在装有一毫升HBSS的冰板上。用无菌手术刀将肾脏切成一毫米的小块。将切碎的肾脏与一毫米新鲜制备的II型胶原酶在37摄氏度下孵育35分钟，并在水平摇床上轻轻倾斜。消化后，加入四毫升含有10%FBS的RPMI以中和胶原酶。通过无菌的100微米细胞过滤器将消化的组织过滤到50毫升管中。使用无菌 1.5 毫升管的底部将消化的组织搅拌到过滤器上，让肾小球通过。用15毫升HBSS冲洗细胞过滤器。然后，在室温下以200 XG离心流过五分钟。在这个阶段，管子包含三层。上层包含较小的结构，例如小管，中间层是带有肾小球的珠子，底层包含碎片。小心地吸出上清液和顶部白色层。然后，将含有带有肾小球和碎片的珠子的沉淀重悬于1.5毫升HBSS中，并将其转移到两毫升管中。将管放入磁性浓缩器中并吸出上清液，然后用一毫升HBSS洗涤珠子两次。将20微升细胞悬液放在载玻片上。然后，在显微镜下观察载玻片是否存在肾小球。接下来，将沉淀重悬于内皮细胞生长培养基中，并将悬浮液转移到两毫升管中。每三毫升培养基加入一微升干扰素γ，并将细胞置于六孔板中，在33摄氏度下孵育。培养三天后，小心地用新鲜培养基替换一毫升培养基。在这个阶段，细胞是异质的，混合了肾小球细胞。七天后，用两毫升新鲜内皮细胞生长培养基替换培养基，补充干扰素γ以开始细胞增殖。10天后，当细胞达到80%汇合时，使用胰蛋白酶传代它们并将其转移到涂有胶原蛋白IV的T-25烧瓶中.培养21天后，将细胞转移到T-75烧瓶中。将三毫升0.25%胰蛋白酶加入T-75烧瓶中，并将细胞在37摄氏度下孵育五分钟。然后，用三毫升内皮生长培养基中和胰蛋白酶，并将悬浮液转移到15毫升管中，以200XG离心五分钟。吸出上清液并在含有1%BSA，2毫摩尔EDTA，1%青霉素/链霉素的一毫升PBS中洗涤细胞。再次，将沉淀离心并重悬于200微升CD31抗体包被的珠子和800微升PBS中。将细胞孵育45分钟，在37摄氏度和5%二氧化碳下连续振荡。接下来，将试管放入磁浓缩器中，并用含有BSA，EDTA和青霉素/链霉素的PBS洗涤细胞四次。最后一次洗涤后，将细胞重悬于三毫升内皮生长培养基中，补充有干扰素γ。在六孔板上的胶原蛋白IV包被孔中每孔板1.5毫升细胞，并在33摄氏度的许可条件下培养。四天后，用新鲜的内皮生长培养基替换750微升培养基，并补充干扰素γ。培养10至14天后，使用带有488纳米过滤器的落射荧光显微镜，观察生长的CD31阳性肾小球内皮细胞或GECs，表达荧光线粒体的集落。21天后，或当细胞达到80%至90%汇合时，将它们转移到T-25烧瓶中。用含有10%FCS的RPMI生长培养基维持培养物。或者，细胞可以冷冻保存。如前所述，使用胰蛋白酶传代细胞。然后，离心细胞并将沉淀重悬于三毫升冷冻培养基中。将细胞分装在冷冻管中，并将其储存在液氮，蒸气温度下。该协议描述了一种分离肾小球内皮细胞的方法。从分离的肾小球开始，细胞在培养三天后开始缓慢生长。七天后，细胞出现异质性，并显示其他肾小球细胞类型，如足细胞、壁细胞、上皮细胞和系膜细胞。在达到70%至80%汇合度后，使用阳性选择的内皮细胞去除其他肾小球细胞。MitoDendra2 GEC表达CD31并且足细胞标志物呈阴性，如突触足素阴性染色所示。检查葡萄糖浓度对线粒体结构的影响。与正常葡萄糖下细胞中可见的细长线粒体相比，高葡萄糖诱导线粒体碎裂或裂变，如突出的球状线粒体所观察到的那样。此外，使用405纳米激光在单个活的MitoDendra2 GEC中光转换选定的线粒体亚群。显示了线粒体在正常和高葡萄糖浓度处理细胞的选定区域中从绿色到红色的成功光转换。这种切换允许见证MitoDendra2 GEC中的聚变事件。在正常葡萄糖下，由于线粒体基质融合，观察到黄色合并的绿色和红色荧光。相反，在高葡萄糖处理的GEC中，线粒体主要是碎片化的。将细胞放置在一个带有少量生长培养基的六孔板中对于让细胞附着在培养皿上至关重要。分离的内皮细胞可用于进行线粒体功能和结构研究。使用具有荧光线粒体特征的永生化内皮细胞将为药物发现铺平道路。可以在细胞上测试小分子，并进行实时成像以检查它们对线粒体功能的影响。

Research

JoVE Journal

Biology

Isolation of Human Umbilical Vein Endothelial Cells (HUVEC)

Isolation of Human Umbilical Vein Endothelial Cells HUVEC

分离人脐静脉内皮细胞（HUVEC）

Angiogenesis is a complex multi-step process, where in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps ...

科研

生物学

Isolation of Valvular Endothelial Cells

瓣膜内皮细胞的分离

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system.  These thin, fibrous tissues are ...

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

从新生小鼠的肺血管内皮细胞的分离和培养

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Isolation of Embryonic Ventricular Endothelial Cells

胚胎心室血管内皮细胞的分离

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines ...

Isolation of Microvascular Endothelial Tubes from Mouse Resistance Arteries

从鼠标阻力动脉分离微血管内皮管

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic ...

Isolation of mRNAs Associated with Yeast Mitochondria to Study Mechanisms of Localized Translation

Most of mitochondrial proteins are encoded in the nucleus and need to be imported into the organelle. Import may occur while the protein is synthesized ...

Isolation of Murine Valve Endothelial Cells

鼠阀门内皮细胞的分离

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

隔离和线粒体从培养细胞和小鼠组织功能分析

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

隔离和鼠标主动脉内皮细胞原代培养

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...