Name: enrichment of mammalian tissues and xenopus oocytes with cholesterol
Uploaded: 2020-03-25T13:00:00
Description: Watch this Scientific Journal Video about Enrichment of Mammalian Tissues and Xenopus Oocytes with Cholesterol at JoVE.com

这项工作得到了美国心脏协会（至A.R.-D.）的科学家发展赠款（11SDG5190025）的支持，以及国家卫生研究所R01授予AA-023764（至A.N.B.）和HL-104631和R37 A-11560（至A.M.D.） 的支持。

田纳西大学健康科学中心（UTHSC）进行了所有动物实验程序。动物护理和实验议定书由UTHSC动物护理和使用委员会审查和批准，该委员会是一个由国际实验室动物护理评估和鉴定协会认可的机构。
1. 使用甲基β-环糊精与胆固醇饱和的组织和细胞浓缩
注：下面的胆固醇浓缩协议适用于组织、细胞和细胞系。例如，我们描述了为丰富哺乳动物脑动脉而执行的步?...

用胆固醇丰富哺乳动物组织和细胞以及Xenopus卵母细胞的方法是研究胆固醇水平升高对单个分子物种、复杂大分子系统（如蛋白质）以及细胞和器官功能的影响的有力工具。在本文中，我们描述了促进此类研究的两种补充方法。首先，我们描述了如何使用富含胆固醇的M+CD来丰富组织和细胞。我们证明，在脑动脉部分，这种方法导致胆固醇水平增加+50%。此外，在最近的一项研究中，我们显示?...

胆固醇是一种主要的细胞脂质，它起着许多关键的功能和结构作用121，2，3，4，5，6，7，8，9。7,8,95,6,,3,4,,从调节血浆膜的物理特性到确保细胞的生存能力、生长、增殖，以及作为信号和前体分子在大量生化途径中，胆固醇是正常细胞和器官功能所必需的必要组成部分。因此，胆固醇缺乏会导致严重的身体畸形和各种疾病。另一方面，即使胆固醇在生理水平（2-3x）以上略有增加，也含有细胞毒性11、2、10，2,10与疾病的发展有关，包括心血管11、12、1312,13和神经退行性疾病1114、15、16、17。14,15,16,17因此，为了询问胆固醇的关键功能并确定胆固醇水平变化的影响，已经开发出了改变组织、细胞和异种卵母细胞中胆固醇含量的不同方法。
哺乳动物组织和细胞中胆固醇水平的变化 有几种方法可以被用来降低组织和细胞18的胆固醇水平。一种方法是接触溶解在脂蛋白缺乏血清中的他汀类药物，以抑制HMG-CoA还原酶，控制胆固醇合成率19，20。19,然而，这些降胆固醇药物也抑制沿美价酮途径形成非固醇产物。因此，加入少量的美价，使这些产品的形成21，并增强这种方法的特异性。降低胆固醇水平的另一种方法是使用β-环糊精。这些糖原蛋白单体单体具有一个内部疏水性腔，其直径与固醇22的大小相匹配，便于从细胞中提取胆固醇，从而消耗其原生胆固醇含量23。例如，2-羟丙基-β-环糊精（HP_CD），这是一种临床前药物，目前正在测试治疗尼曼-皮克C型疾病，一种遗传性致命的代谢紊乱，其特征是淋索质胆固醇储存24。胆固醇消耗水平取决于所使用的特定衍生物。例如，HP_CD 提取的胆固醇的容量低于甲基化衍生物，甲基β-环糊精（M+CD）24，25，26，27，28，29，30。29,3024,25,26,27,28,值得注意的是，然而，β-环糊精还可以提取其他疏水分子，除了胆固醇，这可能会导致非特异性的影响31。与耗竭相比，细胞和组织可以通过与胆固醇23的中饱和的β-环糊精进行治疗，专门与胆固醇一起丰富。这种方法也可用作胆固醇消耗31中使用的β-环糊精特异性的对照。组织和细胞中胆固醇的消耗非常简单，可以通过将细胞暴露30-60分钟到溶解在用于储存细胞的介质中的5 mM M_CD来实现。这种方法可导致胆固醇含量降低50%（例如，在海马神经元32，大鼠脑动脉33）。另一方面，准备β-环糊精-胆固醇复合物用于组织和细胞的胆固醇富集更为复杂，将在协议部分进行说明。
使用饱和胆固醇的β-环糊精来丰富组织和细胞的替代方法涉及使用LDL，它依赖于组织/细胞18中表达的LDL受体。虽然这种方法提供了使用细胞的天然胆固醇平衡机制的优点，但它有几个局限性。首先，不能用这种方法来丰富不表达LDL受体的组织和细胞。其次，低密度脂蛋白颗粒除了胆固醇外，还含有其他脂质。具体来说，LDL由蛋白质ApoB100（25%）组成和以下脂质（75%）：~6-8%胆固醇，+45-50%胆汁酯，+18-24%磷脂，和+4-8%三甘油34。因此，通过低密度脂蛋白颗粒输送胆固醇是非特异性的。第三，表达低密度脂蛋白受体的组织和细胞中低密度脂蛋白增加的百分比可能明显低于使用饱和胆固醇的环糊精观察到的含量。例如，在以前的研究中，通过低密度脂蛋白使啮齿动物脑动脉与胆固醇的丰富率仅使胆固醇水平增加10-15%35。相反，如协议部分所述，这些动脉与胆固醇饱和的动脉的浓缩导致胆固醇含量增加>50%（参见代表结果部分，图1）。
异种卵母细胞中胆固醇水平的变化 异种卵母细胞是一种异质表达系统，通常用于研究细胞和蛋白质功能。早期的研究表明，Xenopus卵母细胞的胆固醇与磷脂摩尔比为0.5± 0.136。由于这种内在的高胆固醇水平，增加胆固醇含量在这个系统中是具有挑战性的，但可以通过膜磷脂和胆固醇制成的分散实现。为此，我们选择的磷脂与用于形成人造平面脂质双层的磷脂相似，包括L-β-磷脂乙醇胺（POPE）和1-棕榈-2-奥莱基-sn-糖二烯-3-磷脂-l-塞林（POPS），如协议部分所述。这种方法可能导致胆固醇含量增加 >50%（参见代表结果部分，图 2）。
用基于磷脂的分散物丰富Xenopus卵母细胞的替代方法是使用胆固醇饱和的环糊精，这与组织和细胞的丰富方式类似。然而，我们发现这种方法的可重复性和效率较低，胆固醇含量平均增加约25%。这可能是由于这两种方法的加载能力不同（请参阅代表结果部分，图 3）。相反，已经表明，使用环糊精来消耗Xenopus卵母细胞的胆固醇，可以导致胆固醇含量降低40%36。
在这里，我们专注于哺乳动物组织和细胞的胆固醇丰富，通过应用环糊精饱和胆固醇，和异种卵母细胞使用脂质体。这两种方法都可以加以利用，以划定胆固醇水平增加对蛋白质功能的影响。蛋白质功能胆固醇调制的机制可能涉及直接相互作用8和/或间接效应9。当胆固醇通过直接相互作用影响蛋白质功能时，胆固醇水平增加对蛋白质活动的影响可能与细胞类型、表达系统或浓缩方法无关。例如，我们利用这两种方法来确定胆固醇对G蛋白的影响，内向纠正钾（GIRK）通道，以心房肌细胞37、海马神经元32、38、HEK29339细胞和异种卵母细胞32,3832、37,37表示。这些研究的结果是一致的：在所有三种类型的哺乳动物细胞和两栖卵母细胞胆固醇上升调节GIRK通道功能（参见代表结果部分，图4，海马神经元和相应的实验在Xenopus卵母细胞）。此外，这些研究中的观察结果也符合在心房肌细胞37，4040和海马神经元32，38,38新鲜从动物接受高胆固醇饮食40的研究的结果一致。37值得注意的是，使用M+CD的海马神经元的胆固醇富集逆转了用于治疗高胆固醇饮食对胆固醇水平和GIRK功能38影响的阿托伐他汀疗法的效果。在其他研究中，我们研究了突变对胆固醇敏感性的影响，使用Xenopus卵母细胞和HEK293细胞41来纠正体内的钾通道Kir2.1。同样，突变对通道灵敏度的影响在两个系统中是相似的。
两种浓缩方法在确定胆固醇水平升高对分子、细胞和器官功能的影响方面应用不计其数。特别是，使用环糊精-胆固醇复合物来丰富细胞和组织是非常普遍的，主要是因为它的特异性。最近的例子包括确定胆固醇对HERG通道激活和底层机制的影响42，发现胆固醇激活G蛋白耦合受体平滑促进刺猪信号43，以及通过膜相关链接蛋白44确定胆固醇在干细胞生物力学中的作用和辅助生成。在我们自己的工作中，我们利用哺乳动物组织富集与M_CD：胆固醇复合物，研究胆固醇富集对基本功能的影响，以及大电导（BK，MaxiK）在血管平滑肌35、45、46中钙和电压门通道（BK，MaxiK）的药理学特征。35,45,46在其他研究中，我们使用基于磷脂的分散方法来丰富具有胆固醇的Xenopus卵母细胞，以确定不同区域在Kir2.1和GIRK通道中胆固醇敏感性41、47、48、4947,48,49中的作用，并确定这些通道4132、50、51中的假定胆固醇结合位,点。32,50

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		<tr>
			<td>Amplex Red Cholesterol Assay Kit</td>
			<td>Invitrogen</td>
			<td>A12216</td>
			<td></td>
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		<tr>
			<td>Pierce BCA Protein Assay Kit</td>
			<td>Thermo Scientific</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>Pre-Diluted Protein Assay Standards BSA set</td>
			<td>Thermo Scientific</td>
			<td>23208</td>
			<td></td>
		</tr>
		<tr>
			<td>Brain PE 25Mg in Chloroform</td>
			<td>Avanti Lipids</td>
			<td>840022C</td>
			<td></td>
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		<tr>
			<td>16:0-18:1 PS 25Mg Chloroform</td>
			<td>Avanti Lipids</td>
			<td>840034C</td>
			<td></td>
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		<tr>
			<td>Cholesterol 100Mg Powder</td>
			<td>Sigma</td>
			<td>C8667</td>
			<td></td>
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			<td>KCl</td>
			<td>Fisher</td>
			<td>P217</td>
			<td></td>
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		<tr>
			<td>Trizma base</td>
			<td>Sigma</td>
			<td>T6066</td>
			<td></td>
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			<td>HEPES</td>
			<td>Corning</td>
			<td>61-034-RO</td>
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			<td>MgCl2</td>
			<td>Fisher</td>
			<td>M33</td>
			<td></td>
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			<td>NaCl</td>
			<td>Fisher</td>
			<td>S271</td>
			<td></td>
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			<td>KH2PO4</td>
			<td>Fisher</td>
			<td>P285</td>
			<td></td>
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			<td>MgSO4</td>
			<td>EMD Chemicals</td>
			<td>MX0070-1</td>
			<td></td>
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			<td>EDTA</td>
			<td>VWR</td>
			<td>E177</td>
			<td></td>
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			<td>Dextrose Anhydrous</td>
			<td>Fisher</td>
			<td>BP350</td>
			<td></td>
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			<td>NaHCO3</td>
			<td>Sigma</td>
			<td>S6014</td>
			<td></td>
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			<td>CaCl2</td>
			<td>Sigma</td>
			<td>C3881</td>
			<td></td>
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			<td>Blood Gas Tank</td>
			<td>nexAir</td>
			<td></td>
			<td></td>
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			<td>NaOH</td>
			<td>Fisher</td>
			<td>S318</td>
			<td></td>
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			<td>1.5mL tubes</td>
			<td>Fisher</td>
			<td>S35818</td>
			<td></td>
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			<td>Gastight Syringe 100uL</td>
			<td>Hamilton</td>
			<td>1710</td>
			<td></td>
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		<tr>
			<td>Microliter Syringe 25uL</td>
			<td>Hamilton</td>
			<td>702</td>
			<td></td>
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		<tr>
			<td>12 mL heavy duty conical centrifuge beaded rim tube</td>
			<td>Pyrex</td>
			<td>8120-12</td>
			<td></td>
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		<tr>
			<td>Chloroform</td>
			<td>Fisher</td>
			<td>C298</td>
			<td></td>
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		<tr>
			<td>Support Stand</td>
			<td>Homescience Tools</td>
			<td>CE-STAN5X8</td>
			<td></td>
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		<tr>
			<td>Universal Clamp, 3-Prong</td>
			<td>Homescience Tools</td>
			<td>CE-CLPUNIV</td>
			<td></td>
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		<tr>
			<td>Sonicator</td>
			<td>Laboratory Supplies</td>
			<td>G112SP1G</td>
			<td></td>
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			<td>3D rotator mixer</td>
			<td>Benchmark Scientific</td>
			<td>B3D 1308</td>
			<td></td>
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		<tr>
			<td>96 well plate</td>
			<td>Sigma</td>
			<td>BR781602</td>
			<td></td>
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		<tr>
			<td>N2 gas</td>
			<td>nexAir</td>
			<td></td>
			<td></td>
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		<tr>
			<td>Glass beakers 40ml-1L</td>
			<td>Fisher</td>
			<td>02-540</td>
			<td></td>
		</tr>
		<tr>
			<td>Ice Machine</td>
			<td>Scotsman</td>
			<td>CU1526MA-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Ice bucket</td>
			<td>Fisher</td>
			<td>50-136-7764</td>
			<td></td>
		</tr>
		<tr>
			<td>1X PBS</td>
			<td>Corning</td>
			<td>21-031-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>TritonX</td>
			<td>Fisher</td>
			<td>BP151-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonic Dismembrator</td>
			<td>Fisher</td>
			<td>Model 100</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf microcentrifuge</td>
			<td>Eppendorf</td>
			<td>Model 5417R</td>
			<td></td>
		</tr>
		<tr>
			<td>Amber bottles</td>
			<td>Fisher</td>
			<td>03-251-420</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning&#8482; Disposable Glass Pasteur Pipets</td>
			<td>FIsher</td>
			<td>13-678-4A</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>FIsher</td>
			<td>50-998-944</td>
			<td></td>
		</tr>
		<tr>
			<td>Isotemp&#8482; BOD Refrigerated Incubator</td>
			<td>FIsher</td>
			<td>97-990E</td>
			<td></td>
		</tr>
		<tr>
			<td>Oocytes</td>
			<td>Xenoocyte&#8482;</td>
			<td>10005</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat</td>
			<td>Envigo</td>
			<td>Sprague Dawley</td>
			<td>weight 250g</td>
		</tr>
		<tr>
			<td>Methyl-&#946;-cyclodextrin</td>
			<td>Sigma</td>
			<td>C4555</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath incubator with shaker</td>
			<td>Precision</td>
			<td>51221080</td>
			<td>Lowest shaker setting O/N 37 &#176;C</td>
		</tr>
		<tr>
			<td>Filipin</td>
			<td>Sigma</td>
			<td>SAE0088-1ML</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Fisher</td>
			<td>BP231</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde 4%</td>
			<td>Mallinckrodt</td>
			<td>2621</td>
			<td></td>
		</tr>
		<tr>
			<td>DI H2O</td>
			<td>University DI source</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold antifade reagnet</td>
			<td>Invitrogen</td>
			<td>P10144</td>
			<td></td>
		</tr>
		<tr>
			<td>Microslides 75x25mm Frosted</td>
			<td>Diagger</td>
			<td>G15978A</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fine Science Tools</td>
			<td>11255-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Coverslip</td>
			<td>Diagger</td>
			<td>G15972B</td>
			<td></td>
		</tr>
		<tr>
			<td>Clear nail polish</td>
			<td>Revlon</td>
			<td>771 Clear</td>
			<td></td>
		</tr>
		<tr>
			<td>Labeling Tape</td>
			<td>Fisher</td>
			<td>15-901-20F</td>
			<td></td>
		</tr>
		<tr>
			<td>Securline Lab Marker II</td>
			<td>Sigma</td>
			<td>Z648205-5EA</td>
			<td></td>
		</tr>
		<tr>
			<td>BD 10mL Syringe</td>
			<td>Fisher</td>
			<td>14-823-16E</td>
			<td></td>
		</tr>
		<tr>
			<td>1.2 &#956;m syringe filter</td>
			<td>VWR</td>
			<td>28150-958</td>
			<td></td>
		</tr>
		<tr>
			<td>KimWipes</td>
			<td>Fisher</td>
			<td>06-666A</td>
			<td></td>
		</tr>
		<tr>
			<td>pH probe</td>
			<td>Sartorus</td>
			<td>py-p112s</td>
			<td></td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td>Denver instrument</td>
			<td>Model 225</td>
			<td></td>
		</tr>
		<tr>
			<td>70% ETOH</td>
			<td>Pharmco</td>
			<td>211USP/NF</td>
			<td></td>
		</tr>
		<tr>
			<td>Timer</td>
			<td>Fisher</td>
			<td>02-261-840</td>
			<td></td>
		</tr>
		<tr>
			<td>Steno book</td>
			<td>Staples</td>
			<td>163485</td>
			<td></td>
		</tr>
	</tbody>
</table>

enrichment of mammalian tissues and xenopus oocytes with cholesterol

哺乳动物组织和细胞的胆固醇丰富，包括用于研究细胞功能的Xenopus卵母细胞，可以使用多种方法完成。在这里，我们描述了用于此目的的两种重要方法。首先，我们描述了如何使用充满胆固醇的环糊精来丰富组织和细胞，以脑动脉（组织）和海马神经元（细胞）为例。此方法可用于任何类型的组织、细胞或细胞系。胆固醇富集的替代方法包括使用低密度脂蛋白（LDL）。这种方法的优点是，它使用部分天然胆固醇平衡机制的细胞。然而，虽然环糊精方法可以应用于丰富任何细胞类型与胆固醇的兴趣，LDL方法仅限于表达LDL受体的细胞（例如，肝细胞，骨髓衍生细胞，如血白细胞和组织巨噬细胞），和浓缩水平取决于LDL受体的浓度和流动性。此外，低密度脂蛋白颗粒包括其他脂质，因此胆固醇的输送是非特异性的。其次，我们描述了如何使用含有胆固醇的磷脂色（即脂质体）来丰富异种卵母细胞的胆固醇。异种卵母细胞是一种流行的异质表达系统，用于研究细胞和蛋白质功能。对于哺乳动物组织（脑动脉）的环糊精胆固醇增生方法，以及Xenopus卵母细胞的磷脂胆固醇浓缩方法，我们证明胆固醇水平在孵育5分钟后达到最大值。在长期潜伏期（例如60分钟），这种胆固醇水平保持不变。这些数据共同为优化组织、细胞和Xenopus卵母细胞的胆固醇富集时间条件提供了基础，用于旨在研究胆固醇富集的影响的功能研究。

使用饱和胆固醇的环糊精作为丰富组织和细胞胆固醇的手段是公认的。在这里，我们首先演示了这种广泛使用的方法的应用，用M+CD饱和胆固醇来丰富大鼠脑动脉的胆固醇。图1A显示了一个图像脑动脉平滑肌肉层的例子，并演示了在组织富集后获得的与菲律宾相关的荧光的浓度依赖性增加，胆固醇浓度从6.25 μM-6.25 mM开始1小时。 <strong class=...

Watch this Scientific Journal Video about Enrichment of Mammalian Tissues and Xenopus Oocytes with Cholesterol at JoVE.com

Enrichment of Mammalian Tissues and Xenopus Oocytes with Cholesterol

提出了两种胆固醇富集方法：应用富含胆固醇的环糊精来丰富哺乳动物组织和细胞，以及利用富含胆固醇的磷脂型分散物（脂质体）来丰富Xenopus卵母细胞。这些方法有助于确定胆固醇水平升高对分子、细胞和器官功能的影响。

哺乳动物组织和异种卵母细胞与胆固醇的丰富

Cholesterol enrichment of mammalian tissues and cells, including Xenopus oocytes used for studying cell function, can be accomplished using a variety of ...

胆固醇升高是心血管和神经退行性疾病的主要危险因素。我们的协议为研究高胆固醇血症的生理和机械后果提供了宝贵的工具。这些程序可以使用基本的实验室设备执行，并适用于细胞、组织和异形细胞。胆固醇是全身细胞膜的主要成分。这些技术可用于研究任何细胞类型中胆固醇水平升高的影响。解剖动脉是最难的一步。小心地从大脑中切除动脉，不要拉伸或损坏血管组织。脂质是非常微妙的，与他们合作更多的是一门艺术，而不是一门科学。视频演示提供了一个指南，用于学习如何小心处理脂质。要准备胆固醇饱和甲基-β-环糊精溶液，首先将064克甲基-β-环糊精加入10毫升PBS，用搅拌棒搅拌溶液，确保甲基-β-环糊精完全溶解。接下来，在烧瓶中加入0024克胆固醇粉，用铲子搅拌溶液，以分解尽可能多的胆固醇块。当几乎所有的胆固醇都溶解后，用至少两层石蜡膜密封烧瓶。在37摄氏度的水浴中，每分钟摇动约30次振荡的烧瓶。8至16小时后，将溶液冷却至室温，然后通过22微米聚氨酯纤维素注射器过滤器将混合物过滤到一类瓶中。对于哺乳动物脑动脉富集，将大脑从250克至300克Sprague Dawley大鼠收获到冰上PBS的烧杯中，然后将大脑转移到一个打蜡的解剖碗中，在解剖显微镜下，含有足够的PBS，使组织陷入水。用两到三个针针固定大脑，确保它们不会穿透血管。并使用锋利的钳子和小手术剪刀轻轻地解剖脑动脉及其分支，形成大脑底部的威利斯圈。接下来，在PBS中短暂冲洗长达一厘米的动脉段，然后用足够的胆固醇富集将冲洗过的动脉段孵育，以覆盖组织。要确定胆固醇水平的任何变化，使用一瓶新鲜的菲利平粉，以二甲基硫化物的染料每毫升10毫克的库存溶液，用三个五分钟的新鲜PBS洗涤胆固醇丰富的组织。上次洗涤后，将动脉段在 4%对甲醛中固定 15 分钟，在防光下，在 PBS 中 5%Triton 中渗透样品 10 分钟。在孵化结束时，在摇床上用PBS洗涤三个五分钟，然后将样品加入每毫升25微克的菲利平染料溶液最终浓度25微克，在室温下洗涤一小时，防止光线。在孵化结束时，按照演示，将动脉片洗三次，然后用蒸馏水短暂冲洗。最后一次洗涤后，使用实验室组织吸收任何多余的液体，并使用适当的安装介质将动脉安装到幻灯片上。小心地用盖滑盖住动脉，注意避免组织滚动或扭曲，让滑轨在室温下干燥24小时，防止光线。当安装介质干燥时，用透明指甲油密封盖滑边，让抛光剂干燥 10 至 15 分钟。然后，用340至380纳米的激发和385至470纳米的发射图像组织。为了准备基于磷脂的分散，将200微升每毫升10毫克氯仿溶解脂质溶液、L-α-磷脂乙醇胺、1-棕榈-2-奥莱霉素-糖-3磷管和胆固醇在12毫升玻璃管中。在氮流下蒸发机罩中的氯仿，然后悬浮在缓冲的 150 毫摩尔氯化钾和 10 毫摩尔 Tris HEPES 溶液的 800 微升中悬浮脂质。然后用石蜡膜密封管，以80千赫轻轻声波化管内色10分钟，直到形成乳白色混合物。对于富含Xenopus卵母细胞的胆固醇，使用锋利的钳子在多个区域破坏雌性Xenopus laevis青蛙的卵巢囊，将卵巢块放入60毫米的盘子里，其中5毫升不含钙ND96，每毫升胶原蛋白补充5毫克。在室温下以每分钟 60 次振荡摇动轨道摇床上的组织 15 分钟。在孵育结束时，使用具有宽尖端的转移移液器，大力移液含有溶液的卵母细胞，以分离单个卵母细胞，并用新鲜无钙ND96快速冲洗暗卵母细胞溶液，直到溶液变得透明。接下来，将90微升的胆固醇富磷脂为基础的分散物转移到96井板的一个井中，并用尽可能少的介质将6个卵母细胞加到井中。将 96 井板放在三维平台旋转器上 5 到 10 分钟。然后向井中加入一滴ND96，并将富含胆固醇的卵母细胞转移到含有ND96的35毫米板中，供其立即分析。在这里，一个图像脑动脉平滑肌肉层的例子表明，在组织富集上获得的脂肪相关荧光的浓度依赖性增加与胆固醇浓度的增加有关。值得注意的是，在甲基-β-环糊精胆固醇复合物治疗后三个小时，胆固醇水平比浓缩后立即降低约50%。虽然一个小时的孵育时间通常用于丰富组织和细胞与胆固醇在这种方法，5分钟的孵化通常足以实现统计显著增加脑动脉胆固醇含量由胆固醇氧化酶为基础的生化测定。虽然 Xenopus 卵母细胞的胆固醇水平没有显著变化，但缺乏胆固醇的基于控制磷脂的分散体，在治疗仅五分钟后，胆固醇水平就显著增加，而包括胆固醇的磷脂为基础的分散体，当孵化时间增加到 60 分钟时，胆固醇水平会显著升高。从海马体的CA1区域新鲜分离的神经元中也证明了基于环糊精的富细胞方法的有效性。事实上，在含有胆固醇的甲基-β-环糊精中孵育神经元60分钟，使胆固醇水平增加两倍以上，如菲律宾相关荧光评估。总的来说，这些程序的两个最重要的步骤是准备胆固醇富集混合物和确保动脉组织的完整性。在胆固醇富集之后，可以研究受高胆固醇血症影响的蛋白质的功能。富含胆固醇细胞的能力有助于研究心肌细胞和神经元中胆固醇水平升高。卵母细胞的使用使我们能够研究胆固醇如何与离子通道结合。一件实验室外套和手套，她总是为这个协议穿。氯仿和甲醛应在烟罩下使用，并丢弃为危险废物。

enrichment-of-mammalian-tissues-and-xenopus-oocytes-with-cholesterol

Research

JoVE Journal

Biochemistry

Enrichment of Mammalian Tissues and Xenopus Oocytes with Cholesterol

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this technique is also frequently referred to as immunoprecipitation. Affinity capture can be applied in a bench-scale and in a high-throughput context. When coupled with protein mass spectrometry, affinity capture has proven to be a workhorse of interactome analysis. Although there are potentially many ways to execute the numerous steps involved, the following protocols implement our favored methods. Two features are distinctive: the use of cryomilled cell powder to produce cell extracts, and antibody-coupled paramagnetic beads as the affinity medium. In many cases, we have obtained superior results to those obtained with more conventional affinity capture practices. Cryomilling avoids numerous problems associated with other forms of cell breakage. It provides efficient breakage of the material, while avoiding denaturation issues associated with heating or foaming. It retains the native protein concentration up to the point of extraction, mitigating macromolecular dissociation. It reduces the time extracted proteins spend in solution, limiting deleterious enzymatic activities, and it may reduce the non-specific adsorption of proteins by the affinity medium. Micron-scale magnetic affinity media have become more commonplace over the last several years, increasingly replacing the traditional agarose- and Sepharose-based media. Primary benefits of magnetic media include typically lower non-specific protein adsorption; no size exclusion limit because protein complex binding occurs on the bead surface rather than within pores; and ease of manipulation and handling using magnets.

Here we describe protocols to disrupt mammalian cells by solid-state milling at a cryogenic temperature, produce a cell extract from the resulting cell powder, and isolate protein complexes of interest by affinity capture upon antibody-coupled micron-scale paramagnetic beads.

从Cryomilled哺乳动物细胞中的蛋白质复合物亲和捕获

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this ...

科研

生物化学

Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids

Voltage-Clamp Fluorometry (VCF) has been the technique of choice to investigate the structure and function of electrogenic membrane proteins where real-time measurements of fluorescence and currents simultaneously report on local rearrangements and global function, respectively1. While high-resolution structural techniques such as cryo-electron microscopy or X-ray crystallography provide static images of the proteins of interest, VCF provides dynamic structural data that allows us to link the structural rearrangements (fluorescence) to dynamic functional data (electrophysiology). Until recently, the thiol-reactive chemistry used for site-directed fluorescent labeling of the proteins restricted the scope of the approach because all accessible cysteines, including endogenous ones, will be labeled. It was thus required to construct proteins free of endogenous cysteines. Labeling was also restricted to sites accessible from the extracellular side. This changed with the use of Fluorescent Unnatural Amino Acids (fUAA) to specifically incorporate a small fluorescent probe in response to stop codon suppression using an orthogonal tRNA and tRNA synthetase pair2. The VCF improvement only requires a two-step injection procedure of DNA injection (tRNA/synthetase pair) followed by RNA/fUAA co-injection. Now, labelling both intracellular and buried sites is possible, and the use of VCF has expanded significantly. The VCF technique thereby becomes attractive for studying a wide range of proteins and &#8211; more importantly &#8211; allows investigating numerous cytosolic regulatory mechanisms.

Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids

This article describes an enhancement of conventional Voltage-Clamp Fluorometry (VCF) where Fluorescent Unnatural Amino Acids (fUAA) are used instead of maleimide dyes, to probe structural rearrangements in ion channels. The procedure includes Xenopus oocyte DNA injection, RNA/fUAA coinjection, and simultaneous current and fluorescence measurements.

电压钳荧光测定<em&gt;爪蟾</em&gt;使用荧光非天然氨基酸的卵母细胞

Voltage-Clamp Fluorometry (VCF) has been the technique of choice to investigate the structure and function of electrogenic membrane proteins where ...

Enrichment of Detergent-insoluble Protein Aggregates from Human Postmortem Brain

In this study, we describe an abbreviated single-step fractionation protocol for the enrichment of detergent-insoluble protein aggregates from human postmortem brain. The ionic detergent N-lauryl-sarcosine (sarkosyl) effectively solubilizes natively folded proteins in brain tissue allowing the enrichment of detergent-insoluble protein aggregates from a wide range of neurodegenerative proteinopathies, such as Alzheimer&#39;s disease (AD), Parkinson&#39;s disease and amyotrophic lateral sclerosis, and prion diseases. Human control and AD postmortem brain tissues were homogenized and sedimented by ultracentrifugation in the presence of sarkosyl to enrich detergent-insoluble protein aggregates including pathologic phosphorylated tau, the core component of neurofibrillary tangles in AD. Western blotting demonstrated the differential solubility of aggregated phosphorylated-tau and the detergent-soluble protein, Early Endosome Antigen 1 (EEA1) in control and AD brain. Proteomic analysis also revealed enrichment of &#946;-amyloid (A&#946;), tau, snRNP70 (U1-70K), and apolipoprotein E (APOE) in the sarkosyl-insoluble fractions of AD brain compared to those of control, consistent with previous tissue fractionation strategies. Thus, this simple enrichment protocol is ideal for a wide range of experimental applications ranging from Western blotting and functional protein co-aggregation assays to mass spectrometry-based proteomics.

An abbreviated fractionation protocol for the enrichment of detergent-insoluble protein aggregates from human postmortem brain is described.

人死后脑中洗涤剂不溶性蛋白的富集

In this study, we describe an abbreviated single-step fractionation protocol for the enrichment of detergent-insoluble protein aggregates from human ...

Imaging Amyloid Tissues Stained with Luminescent Conjugated Oligothiophenes by Hyperspectral Confocal Microscopy and Fluorescence Lifetime Imaging

Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find neurodegenerative diseases such as Alzheimer&#39;s and Parkinson&#39;s disease afflicting primarily the central nervous system, and systemic amyloidosis where serum amyloid A, transthyretin and IgG light chains deposit as amyloid in liver, carpal tunnel, spleen, kidney, heart, and other peripheral tissues. Amyloid has been known and studied for more than a century, often using amyloid specific dyes such as Congo red and Thioflavin T (ThT) or Thioflavin (ThS). In this paper, we present heptamer-formyl thiophene acetic acid (hFTAA) as an example of recently developed complements to these dyes called luminescent conjugated oligothiophenes (LCOs). hFTAA is easy to use and is compatible with co-staining in immunofluorescence or with other cellular markers. Extensive research has proven that hFTAA detects a wider range of disease associated protein aggregates than conventional amyloid dyes. In addition, hFTAA can also be applied for optical assignment of distinct aggregated morphotypes to allow studies of amyloid fibril polymorphism. While the imaging methodology applied is optional, we here demonstrate hyperspectral imaging (HIS), laser scanning confocal microscopy and fluorescence lifetime imaging (FLIM). These examples show some of the imaging techniques where LCOs can be used as tools to gain more detailed knowledge of the formation and structural properties of amyloids. An important limitation to the technique is, as for all conventional optical microscopy techniques, the requirement for microscopic size of aggregates to allow detection. Furthermore, the aggregate should comprise a repetitive &#946;-sheet structure to allow for hFTAA binding. Excessive fixation and/or epitope exposure that modify the aggregate structure or conformation can render poor hFTAA binding and hence pose limitations to accurate imaging.

Amyloid deposition is a hallmark of different diseases and afflicts many different organs. This paper describes the application of luminescent conjugated oligothiophene fluorescence staining in combination with fluorescence microscopy techniques. This staining method represents a powerful tool for detection and exploration of protein aggregates in both clinical and scientific setups.

用高光谱共聚焦显微镜和荧光寿命成像对发光共轭吩染色的淀粉样组织的成像

Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find ...

Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation of protein synthesis is the key to cellular stress-response, and dysregulation is central to many disease states, such as cancer. For instance, although cellular stress leads to the inhibition of global translation by attenuating cap-dependent initiation, certain stress-response proteins are selectively translated in a cap-independent manner. Discreet RNA regulatory elements, such as cellular internal ribosome entry sites (IRESes), allow for the translation of these specific mRNAs. Identification of such mRNAs, and the characterization of their regulatory mechanisms, have been a key area in molecular biology. Toeprinting is a method for the study of RNA structure and function as it pertains to translation initiation. The goal of toeprinting is to assess the ability of in vitro transcribed RNA to form stable complexes with ribosomes under a variety of conditions, in order to determine which sequences, structural elements, or accessory factors are involved in ribosome binding&#8212;a pre-cursor for efficient translation initiation. Alongside other techniques, such as western analysis and polysome profiling, toeprinting allows for a robust characterization of mechanisms for the regulation of translation initiation.

Toeprinting aims to measure the ability of in vitro transcribed RNA to form translation initiation complexes with ribosomes under a variety of conditions. This protocol describes a method for toeprinting mammalian RNA and can be used to study both cap-dependent and IRES-driven translation.

哺乳动物基因翻译起始复合体形成的 Toeprinting 分析

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation ...

Expression and Purification of Mammalian Bestrophin Ion Channels

The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca2+-activated Cl- channel (CaCC) predominantly expressed in retinal pigment epithelium (RPE). The physiological and pathological significance of BEST1 is highlighted by the fact that over 200 distinct mutations in the BEST1 gene have been genetically linked to a spectrum of at least five retinal degenerative disorders, such as Best vitelliform macular dystrophy (Best disease). Therefore, understanding the biophysics of bestrophin channels at the single-molecule level holds tremendous significance. However, obtaining purified mammalian ion channels is often a challenging task. Here, we report a protocol for the expression of mammalian bestrophin proteins with the BacMam baculovirus gene transfer system and their purification by affinity and size-exclusion chromatography. The purified proteins have the potential to be utilized in subsequent functional and structural analyses, such as electrophysiological recording in lipid bilayers and crystallography. Importantly, this pipeline can be adapted to study the functions and structures of other ion channels.

The purification of ion channels is often challenging, but once achieved, it can potentially allow in vitro investigations of the functions and structures of the channels. Here, we describe the stepwise procedures for the expression and purification of mammalian bestrophin proteins, a family of Ca2+-activated Cl- channels.

哺乳动物 Bestrophin 离子通道的表达与纯化

The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca2+-activated Cl- ...

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ubiquitinated proteins, peptides with a diglycine remnant conjugated to the epsilon amino group of lysine (&#39;K-&#949;-diglycine&#39; or simply &#39;diGly&#39;) can be used to track back the original modification site. Efficient immunopurification of diGly peptides combined with sensitive detection by mass spectrometry has resulted in a huge increase in the number of ubiquitination sites identified up to date. We have made several improvements to this workflow, including offline high pH reverse-phase fractionation of peptides prior to the enrichment procedure, and the inclusion of more advanced peptide fragmentation settings in the ion routing multipole. Also, more efficient cleanup of the sample using a filter-based plug in order to retain the antibody beads results in a greater specificity for diGly peptides. These improvements result in the routine detection of more than 23,000 diGly peptides from human cervical cancer cells (HeLa) cell lysates upon proteasome inhibition in the cell. We show the efficacy of this strategy for in-depth analysis of the ubiquitinome profiles of several different cell types and of in vivo samples, such as brain tissue. This study presents an original addition to the toolbox for protein ubiquitination analysis to uncover the deep cellular ubiquitinome.

We present a method for the purification, detection, and identification of diGly peptides that originate from ubiquitinated proteins from complex biological samples. The presented method is reproducible, robust, and outperforms published methods with respect to the level of depth of the ubiquitinome analysis.

通过肽浓缩和质谱检测蛋白质泛化位点检测

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ...

Studying RNA Interactors of Protein Kinase RNA-Activated during the Mammalian Cell Cycle

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral RNAs. When bound to viral double-stranded RNAs (dsRNAs), PKR undergoes dimerization and subsequent autophosphorylation. Phosphorylated PKR (pPKR) becomes active and induces phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2&#945;) to suppress global translation. Increasing evidence suggests that PKR can be activated under physiological conditions such as during the cell cycle or under various stress conditions without infection. However, our understanding of the RNA activators of PKR is limited due to the lack of a standardized experimental method to capture and analyze PKR-interacting dsRNAs. Here, we present an experimental protocol to specifically enrich and analyze PKR bound RNAs during the cell cycle using HeLa cells. We utilize the efficient crosslinking activity of formaldehyde to fix PKR-RNA complexes and isolate them via immunoprecipitation. PKR co-immunoprecipitated RNAs can then be further processed to generate a high-throughput sequencing library. One major class of PKR-interacting cellular dsRNAs is mitochondrial RNAs (mtRNAs), which can exist as intermolecular dsRNAs through complementary interaction between the heavy-strand and the light-strand RNAs. To study the strandedness of these duplex mtRNAs, we also present a protocol for strand-specific qRT-PCR. Our protocol is optimized for the analysis of PKR-bound RNAs, but it can be easily modified to study cellular dsRNAs or RNA-interactors of other dsRNA binding proteins.

We present experimental approaches for studying RNA-interactors of double-stranded RNA binding protein kinase RNA-activated (PKR) during the mammalian cell cycle using HeLa cells. This method utilizes formaldehyde to crosslink RNA-PKR complexes and immunoprecipitation to enrich PKR-bound RNAs. These RNAs can be further analyzed through high-throughput sequencing or qRT-PCR.

哺乳动物细胞周期内蛋白质激酶 rna 激活的 rna 相互作用研究

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral ...

Enrichment of Native Lipoprotein Particles with microRNA and Subsequent Determination of Their Absolute/Relative microRNA Content and Their Cellular Transfer Rate

Lipoprotein particles are predominately transporters of lipids and cholesterol in the bloodstream. Furthermore, they contain small amounts of strands of noncoding microRNA (miRNA). In general, miRNA alters the protein expression profile due to interactions with messenger-RNA (mRNA). Thus, knowledge of the relative and absolute miRNA content of lipoprotein particles is essential to estimate the biological effect of cellular particle uptake. Here, a quantitative real-time polymerase chain reaction (qPCR)-based protocol is presented to determine the absolute miRNA content of lipoprotein particles&#8212;exemplified shown for native and miRNA-enriched lipoprotein particles. The relative miRNA content is quantified using multiwell microfluidic array cards. Furthermore, this protocol allows scientists to estimate the cellular miRNA and, thus, the lipoprotein particle uptake rate. A significant increase of the cellular miRNA level is observable when using high-density lipoprotein (HDL) particles artificially loaded with miRNA, whereas incubation with native HDL particles yields no significant effect due to their rather low miRNA content. In contrast, the cellular uptake of low-density lipoprotein (LDL) particles&#8212;neither with native miRNA nor artificially loaded with it&#8212;did not alter the cellular miRNA level.

Here, a quantitative real-time polymerase chain reaction-based protocol is presented for the determination of the native micro-RNA content (absolute/relative) of lipoprotein particles. In addition, a method for increasing the micro-RNA level, as well as a method for determining the cellular uptake rate of lipoprotein particles, is demonstrated.

微 Rna 对原生脂蛋白颗粒的富集及其与绝对相对微 Rna 含量及细胞转移速率的测定

Lipoprotein particles are predominately transporters of lipids and cholesterol in the bloodstream. Furthermore, they contain small amounts of strands of ...

Quantification of Coenzyme A in Cells and Tissues

Emerging research has revealed that the cellular coenzyme A (CoA) supply can become limiting with a detrimental impact on growth, metabolism and survival. Measurement of cellular CoA is a challenge due to its relatively low abundance and the dynamic conversion of free CoA to CoA thioesters that, in turn, participate in numerous metabolic reactions. A method is described that navigates through potential pitfalls during sample preparation to yield an assay with a broad linear range of detection that is suitable for use in many biomedical laboratories.

This method describes sample preparation from cultured cells and animal tissues, extraction and derivatization of coenzyme A in the samples, followed by high pressure liquid chromatography for purification and quantification of the derivatized coenzyme A by absorbance or fluorescence detection.

细胞和组织中辅酶A的定量

Emerging research has revealed that the cellular coenzyme A (CoA) supply can become limiting with a detrimental impact on growth, metabolism and survival. ...