Name: simultaneous measurements of intracellular calcium and membrane potential in freshly isolated and intact mouse cerebral endothelium
Uploaded: 2019-01-20T14:00:00
Description: Watch this Scientific Journal Video about Simultaneous Measurements of Intracellular Calcium and Membrane Potential in Freshly Isolated and Intact Mouse Cerebral Endothelium at JoVE.com

我们感谢查尔斯·休伊特提供了出色的技术援助, 同时建立了目前协议所需的设备和用品。我们感谢 llu 围产儿生物学中心的 sean m. wilson 博士和 christopher g. wilson 博士分别为我们提供了一个额外的倒置显微镜和静电计。这项研究得到了国家卫生研究院 r00-ag047198 (ejb) 和 loma linda 大学医学院新教师启动资金的支持。内容完全由作者负责, 不一定代表国家卫生研究院的官方观点。

在进行以下实验之前, 请确保所有动物护理使用和协议都得到机构动物护理和使用委员会 (iacuc) 的批准, 并按照国家研究委员会的"动物护理和使用指南" 进行。实验动物" (第8版, 2011) 和 arrive 指南。洛玛·琳达大学的 iacuc 已经批准了这份手稿中用于雄性和雌性 c57bl6 小鼠的所有协议 (年龄范围: 3至30岁)。
1. 设备和材料
请注意:</strong...

<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=Temperature+effects+on+morphological+integrity+and+Ca2++signaling+in+freshly+isolated+murine+feed+artery+endothelial+cell+tubes.">Temperature effects on morphological integrity and Ca2+ signaling in freshly isolated murine feed artery endothelial cell tubes.</a> American Journal of Physiology-Heart and Circulatory Physiology. 301 (3), H773-H783 (2011).</li><li>Socha, M. J., Segal, S. S. <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+microvascular+endothelial+tubes+from+mouse+resistance+arteries.">Isolation of microvascular endothelial tubes from mouse resistance arteries.</a> Journal of Visualized Experiments. (81), (2013).</li><li>Ye, X., Beckett, T., Bagher, P., Garland, C. J., Dora, K. 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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=Membrane+potential+governs+calcium+influx+into+microvascular+endothelium:+integral+role+for+muscarinic+receptor+activation.">Membrane potential governs calcium influx into microvascular endothelium: integral role for muscarinic receptor activation.</a> The Journal of Physiology. 593 (20), 4531-4548 (2015).</li><li>Behringer, E. J., Segal, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+Aging+on+Calcium+Signaling+and+Membrane+Potential+in+Endothelium+of+Resistance+Arteries:+A+Role+for+Mitochondria.">Impact of Aging on Calcium Signaling and Membrane Potential in Endothelium of Resistance Arteries: A Role for Mitochondria.</a> The Journal of Gerontology, Series A: Biological Sciences and Medical Sciences. 72 (12), 1627-1637 (2017).</li><li>Behringer, E. J., 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=Calcium+and+electrical+dynamics+in+lymphatic+endothelium.">Calcium and electrical dynamics in lymphatic endothelium.</a> The Journal of Physiology. 595 (24), 7347-7368 (2017).</li><li>Simmers, M. B., Pryor, A. W., Blackman, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arterial+shear+stress+regulates+endothelial+cell-directed+migration,+polarity,+and+morphology+in+confluent+monolayers.">Arterial shear stress regulates endothelial cell-directed migration, polarity, and morphology in confluent monolayers.</a> American Journal of Physiology-Heart and Circulatory Physiolog. 293 (3), H1937-H1946 (2007).</li><li>Sandow, S. L., Grayson, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limits+of+isolation+and+culture:+intact+vascular+endothelium+and+BKCa.">Limits of isolation and culture: intact vascular endothelium and BKCa.</a> American Journal of Physiology-Heart and Circulatory Physiology. 297 (1), H1-H7 (2009).</li><li>Diaz-Otero, J. M., Garver, H., Fink, G. D., Jackson, W. F., Dorrance, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aging+is+associated+with+changes+to+the+biomechanical+properties+of+the+posterior+cerebral+artery+and+parenchymal+arterioles.">Aging is associated with changes to the biomechanical properties of the posterior cerebral artery and parenchymal arterioles.</a> American Journal of Physiology-Heart and Circulatory Physiology. 310 (3), H365-H375 (2016).</li><li>Kochukov, M. Y., Balasubramanian, A., Abramowitz, J., Birnbaumer, L., Marrelli, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+endothelial+transient+receptor+potential+C3+channel+is+required+for+small+conductance+calcium-activated+potassium+channel+activation+and+sustained+endothelial+hyperpolarization+and+vasodilation+of+cerebral+artery.">Activation of endothelial transient receptor potential C3 channel is required for small conductance calcium-activated potassium channel activation and sustained endothelial hyperpolarization and vasodilation of cerebral artery.</a> Journal of the American Heart Association. 3 (4), (2014).</li><li>Zhang, L., Papadopoulos, P., Hamel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+TRPV4+channels+mediate+dilation+of+cerebral+arteries:+impairment+and+recovery+in+cerebrovascular+pathologies+related+to+Alzheimer's+disease.">Endothelial TRPV4 channels mediate dilation of cerebral arteries: impairment and recovery in cerebrovascular pathologies related to Alzheimer's disease.</a> British Journal of Pharmacology. 170 (3), 661-670 (2013).</li><li>Socha, M. 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根据最近的发展 6,15,16, 17, 我们现在演示的方法, 分离小鼠脑动脉内皮, 准备同时测量 [ca2 +]i和 vm 在37°c 下始终为 ~ 2小时的 edh 基础。虽然技术上困难, 但我们也可以测量细胞间耦合 (参见参考6, 图 1)。通过这种方式, 我们可以同时测量离散和传导信号事件。有...

血管网络1中脑动脉和动脉之间血管舒张的协调调节整个大脑的血液流动.大脑阻力动脉内的内皮细胞根据需要控制血管直径的变化, 以满足神经元1,2,3的代谢需求。特别是在内皮细胞产生的超极化 (俗称 edh) 期间, 细胞内 ca2 + ([ca2 +]i) 和内皮细胞中的电信号键协调内皮细胞之间的血管舒张作用。周围的平滑肌细胞通过缝隙连接动脉放松4。edh 的生理启动顺序意味着刺激 gq偶联受体 (gpcr), 增加 [ca2 +]i, 激活内皮小和中-钙 2 + 激活 k+(skca/ik ca)通道超极化脑血管电位 (vm)5,6,7。因此, 内皮 [ca2 +]i和 vm 之间的亲密关系是血液流动调节的组成部分, 也是心脑血管功能6、8 不可缺少的。在广泛的文献中, 大量的研究报告了血管内皮功能障碍与慢性病的发展 (如高血压, 糖尿病, 心力衰竭, 冠状动脉疾病, 慢性肾功能衰竭,外周动脉疾病)9,10, 表明研究内皮功能在生理和病理条件的意义。
血管内皮是产生超极化、血管扩张和组织灌注的组成部分, 因此, 检查其自身的自身特性至关重要。作为一般的研究模型, 小鼠动脉内皮管模型的制备已发表之前为骨骼肌11,12, 肠 13,肺14, 最近为大脑6。特别是同时进行的 [ca2 +]i和 vm 测量的研究已经发表了骨骼肌动脉内皮 15,16以及淋巴管内皮细胞 17.除了使用内皮管方法的初步研究外, 还可以参考对其优缺点的全面审查, 8 以确定这一实验工具是否适合于特定的研究。简而言之, 一个优点是保留了内皮细胞功能的关键生理成分 (例如, ca2+流入和细胞内释放, vm 的超极化到 nernstk +的潜力通过skca/ikca 激活, 和内皮细胞间耦合通过缝隙连接), 没有混淆因素, 如血管周围神经输入, 平滑肌电压门控通道功能和收缩力,血液循环, 和荷尔蒙的影响8。相反, 常用的细胞培养方法在形态 18和离子通道表达19方面有显著的改变, 这种改变可以极大地模糊与在体内确定的生理观察的比较。在体内。限制包括缺乏与调节血液流动的其他基本成分的整合, 如平滑肌和实验时间表中的灵活性受限, 因为该模型在完整的血管段隔离4小时内进行了最佳测试从动物。
根据 socha 和 segal12编写的前一段视频协议以及在中间6、15、16 的最新实验发展, 我们在此演示了从后脑动脉和同时测量内皮 [ca2 +]i和 v m分别使用 fura-2 分光光度法和细胞内尖锐电极。此外, 这项实验需要在生理条件下 (ph 7.4, 37°c) 持续超融合盐溶液和药理剂。我们选择了大脑后动脉, 因为它产生的是具有结构完整性 (细胞通过缝隙连接结合) 和足够尺寸 (宽度≥50μm, 长度≥300μm) 的孤立内皮, 适合细胞内和细胞间信号。内皮细胞。此外, 啮齿类动物大脑后动脉的研究在文献中得到了大量的体现, 包括对基本内皮信号机制、血管发育和病理20的检查,21,22. 这一实验应用预计将产生对大脑内皮功能 (和功能障碍) 的高通量分析, 从而使整个过程中对血液流动调节的理解有重大进展。衰老和神经退行性疾病的发展。

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			<td>Glucose</td>
			<td>Sigma-Aldrich (St. Louis, MO, USA)</td>
			<td>G7021</td>
			<td></td>
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			<td>NaCl</td>
			<td>Sigma</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M2670</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>223506</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H4034</td>
			<td></td>
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		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9541</td>
			<td></td>
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		<tr>
			<td>NaOH</td>
			<td>Sigma</td>
			<td>S8045</td>
			<td></td>
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			<td>ATP</td>
			<td>Sigma</td>
			<td>A2383</td>
			<td></td>
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			<td>HCl</td>
			<td>ThermoFisher Scientific (Pittsburgh, PA, USA)</td>
			<td>A466250</td>
			<td></td>
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			<td>Collagenase (Type H Blend)</td>
			<td>Sigma</td>
			<td>C8051</td>
			<td></td>
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		<tr>
			<td>Dithioerythritol</td>
			<td>Sigma</td>
			<td>D8255</td>
			<td></td>
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		<tr>
			<td>Papain</td>
			<td>Sigma</td>
			<td>P4762</td>
			<td></td>
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		<tr>
			<td>Elastase</td>
			<td>Sigma</td>
			<td>E7885</td>
			<td></td>
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			<td>BSA</td>
			<td>Sigma</td>
			<td>A7906</td>
			<td></td>
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		<tr>
			<td>Propidium iodide</td>
			<td>Sigma</td>
			<td>P4170</td>
			<td></td>
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			<td>DMSO</td>
			<td>Sigma</td>
			<td>D8418</td>
			<td></td>
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		<tr>
			<td>Fura-2 AM dye</td>
			<td>Invitrogen, Carlsbad, CA, USA</td>
			<td>F14185</td>
			<td></td>
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			<td>Recirculating chiller (Isotemp 500LCU)</td>
			<td>ThermoFisher Scientific</td>
			<td>13874647</td>
			<td></td>
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			<td>Plexiglas superfusion chamber&#160;</td>
			<td>Warner Instruments, Camden, CT, USA</td>
			<td>RC-27</td>
			<td></td>
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			<td>Glass coverslip bottom (2.4&#160;&#215;&#160;5.0&#160;cm)</td>
			<td>ThermoFisher Scientific</td>
			<td>12-548-5M</td>
			<td></td>
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			<td>Anodized aluminum platform (diameter: 7.8&#160;cm)&#160;</td>
			<td>Warner Instruments</td>
			<td>PM6 or PH6</td>
			<td></td>
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			<td>Compact aluminum stage&#160;</td>
			<td>Siskiyou, Grants Pass, OR, USA</td>
			<td>8090P</td>
			<td></td>
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		<tr>
			<td>Micromanipulator</td>
			<td>Siskiyou</td>
			<td>&#160;MX10</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscopes&#160;</td>
			<td>Zeiss, NY, USA</td>
			<td>Stemi 2000 &#38; 2000-C</td>
			<td></td>
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		<tr>
			<td>Fiber optic light sources</td>
			<td>&#160;Schott, Mainz, Germany &#38; KL200, Zeiss</td>
			<td>Fostec 8375</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon inverted microscope</td>
			<td>Nikon Instruments Inc, Melville, NY, USA</td>
			<td>Ts2</td>
			<td></td>
		</tr>
		<tr>
			<td>Phase contrast objectives&#160;</td>
			<td>Nikon Instruments Inc</td>
			<td>&#160;(Ph1 DL; 10X &#38; 20X)</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent objectives&#160;</td>
			<td>Nikon Instruments Inc</td>
			<td>20X (S-Fluor), and 40X (Plan Fluor)</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon inverted microscope</td>
			<td>Nikon Instruments Inc</td>
			<td>Eclipse TS100</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsyringe pump controller (Micro4 )&#160;</td>
			<td>World Precision Instruments (WPI), Sarasota, FL, USA</td>
			<td>SYS-MICRO4</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibration isolation table</td>
			<td>Technical Manufacturing, Peabody, MA, USA</td>
			<td>&#160;Micro-g</td>
			<td></td>
		</tr>
		<tr>
			<td>Amplifiers</td>
			<td>Molecular Devices, Sunnyvale, CA, USA</td>
			<td>Axoclamp 2B &#38; Axoclamp 900A</td>
			<td></td>
		</tr>
		<tr>
			<td>Headstages&#160;</td>
			<td>Molecular Devices</td>
			<td>HS-2A &#38; HS-9A</td>
			<td></td>
		</tr>
		<tr>
			<td>Function generator&#160;</td>
			<td>EZ Digital, Seoul, South Korea</td>
			<td>FG-8002</td>
			<td></td>
		</tr>
		<tr>
			<td>Data Acquision System</td>
			<td>Molecular Devices, Sunnyvale, CA, USA</td>
			<td>Digidata 1550A</td>
			<td></td>
		</tr>
		<tr>
			<td>Audible Baseline Monitors</td>
			<td>Ampol US LLC, Sarasota, FL, USA</td>
			<td>&#160;BM-A-TM</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital Storage Oscilloscope</td>
			<td>Tektronix, Beaverton, Oregon, USA</td>
			<td>&#160;TDS 2024B</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence System Interface, ARC Lamp + Power Supply, Hyperswitch, PMT</td>
			<td>Molecular Devices, Sunnyvale, CA, USA</td>
			<td>IonOptix Systems</td>
			<td></td>
		</tr>
		<tr>
			<td>Temperature Controller&#160;&#160;</td>
			<td>Warner Instruments</td>
			<td>TC-344B or C</td>
			<td></td>
		</tr>
		<tr>
			<td>Inline Heater&#160;</td>
			<td>Warner Instruments</td>
			<td>SH- 27B</td>
			<td></td>
		</tr>
		<tr>
			<td>Valve Controller&#160;</td>
			<td>Warner Instruments</td>
			<td>VC-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Inline Flow Control Valve</td>
			<td>Warner Instruments</td>
			<td>&#160;FR-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Electronic Puller&#160;</td>
			<td>Sutter Instruments, Novato, CA, USA</td>
			<td>P-97 or P-1000&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Microforge</td>
			<td>Narishige, East Meadow, NY, USA</td>
			<td>&#160;MF-900</td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate Glass Tubes (Trituration)</td>
			<td>World Precision Instruments (WPI), Sarasota, FL, USA</td>
			<td>1B100-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate Glass Tubes (Pinning)</td>
			<td>Warner Instruments</td>
			<td>G150T-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate Glass Tubes (Sharp Electrodes)</td>
			<td>Warner Instruments</td>
			<td>GC100F-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe Filter (0.22 &#181;m)&#160;&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>722-2520</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Petri Dish + Charcoal Sylgard</td>
			<td>Living Systems Instrumentation, St. Albans City, VT, USA</td>
			<td>DD-90-S-BLK</td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas Style Scissors (3 mm &#38; 9.5&#160;mm)</td>
			<td>World Precision Instruments</td>
			<td>555640S, 14364</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors 3 &#38; 7 mm blades</td>
			<td>Fine Science Tools (or FST), Foster City, CA, USA</td>
			<td>Moria MC52 &#38; 15000-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Sharpened fine-tipped forceps&#160;</td>
			<td>FST</td>
			<td>Dumont #5 &#38; Dumont #55</td>
			<td></td>
		</tr>
	</tbody>
</table>

simultaneous measurements of intracellular calcium and membrane potential in freshly isolated and intact mouse cerebral endothelium

大脑动脉及其各自的微循环通过血液流动调节向大脑输送氧气和营养物质。内皮细胞排列血管腔, 并根据需要控制血管直径的变化, 以满足神经元的代谢需求。膜电位 (vm) 和一氧化氮超极化的初级内皮依赖性信号通路通常平行调节血管扩张, 从而增加血液流动。虽然在几个毫米的血管长度的血管扩张协调不可或缺的一部分, 内皮衍生的超极化 (edh) 的组成部分一直是很难测量的历史。edh 的这些成分包含细胞内 ca2 + [ca2 +]i增加并随后激活小型和中间电导 ca2 +-激活 k + (skca/ik ca)通道.
在这里, 我们提出了一个从小鼠大脑动脉中分离新鲜内皮细胞的简化说明;同时测量内皮 [ca2 +] i 和 v m 分别使用 fura-2 分光光度法和细胞内尖锐电极;以及在生理条件下 (ph 7.4, 37°c) 下盐溶液和药理剂的连续超融合。从威利斯圆环的脑后动脉被移除后部沟通和基底动脉。经清理的脑后动脉段的酶消化和随后的三口治疗有助于切除外膜、血管周围神经和平滑肌细胞。然后, 在显微镜下固定下断陷的后脑动脉内皮 "管", 并在连续超融合的情况下使用相机、光电倍增管和一到两个静电计进行检查。总之, 这种方法可以同时测量在离散细胞位置的内皮 [ca2 +]i和 vm 的变化, 除了通过间隙连接传播 edh, 直到毫米的距离沿完整内皮。这种方法有望产生一个高通量分析大脑内皮功能的基础机制的血液流动调节在正常和患病的大脑。

上述协议的示意图如所附数字所示。图 1 a显示了从年轻成年雄性 c57bl/6n 小鼠 (5个月) 中分离出的大脑。脑后动脉从威利斯的圆圈仔细分离, 在没有结缔组织的情况下切除, 并切割成段 (图 1b-d)。从部分消化的动脉段, 完整的内皮管产生, 并固定在玻璃盖滑使用固定移液器。采用两个固定移液器进行温和的机械拉伸, 以?...

Watch this Scientific Journal Video about Simultaneous Measurements of Intracellular Calcium and Membrane Potential in Freshly Isolated and Intact Mouse Cerebral Endothelium at JoVE.com

Simultaneous Measurements of Intracellular Calcium and Membrane Potential in Freshly Isolated and Intact Mouse Cerebral Endothelium

这里演示的是 (1) 新鲜隔离完整的大脑内皮 "管" 和 (2) 同时测量内皮钙和膜电位的协议, 在内皮衍生的超极化过程中。此外, 这些方法允许药理调节内皮细胞钙和电信号作为个人或交互式实验变量。

新鲜分离和内联小鼠脑内皮细胞内钙和膜电位的同时测量

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the ...

该方法有助于回答脑血管生理学领域与慢性病衰老和发育过程中脑血流调节有关的关键问题。该技术的主要优点是，它允许在生理条件下同时测量脑动脉内膜内钙和膜电位。为了隔离老鼠的大脑，在显微镜下去除头骨上的皮肤和头发。并去除过量的血液与冷无钙PSS。接下来，仅使用标准解剖剪刀的尖端进行切口。从腹骨开始，一直延伸到头骨的鼻骨。然后沿着切口小心地打开她的头骨，用粗尖钳子分离结缔组织来暴露大脑。用冷的无钙PSS轻轻清洗分离的大脑，用烧杯去除血液。将脑腹侧放在包含冷解剖溶液的腔室中，用于分离脑动脉。为了隔离脑动脉，通过将两个不锈钢针插入玻璃培养皿底部的木炭注入硅聚合物涂层，将分离的大脑固定在冷解剖溶液中。随后，手术分离后脑动脉约0.3至0.5厘米段从后交流和罗勒动脉。使用不锈钢针脚在 Petri 盘中的解剖溶液中固定两个隔离的后脑动脉。然后，使用锐化细尖钳去除结缔组织，小心地清洁孤立的后脑动脉。将完好无损的动脉切成一到两毫米的段，进行酶消化。在此过程中，使用显微镜、照相机和铝制舞台准备三位一体，以固定腔室和微操纵器。将微丝带与舞台和试样相邻的泵控制器固定。接下来，用矿物油完全回填滴定移液器，并固定在微西林格活塞上。然后使用与泵控制器的微西林格将大约 130 纳米升分离溶液提取到移液器中，同时确保没有气泡。随后，将完好的动脉段放入一毫升分离溶液中，所需浓度的酶放入10毫升玻璃管中。在34摄氏度下孵育10至12分钟，进行部分消化。消化后，用五毫升新鲜分离溶液代替酶溶液。使用一毫升移液器，在室温下将一段转移到含有分离溶液的腔室中。接下来，将移液器放入腔室中的分离溶液中，将其靠近消化容器的一端。在泵控制器上设置 2 到 5 纳米升/秒的速率，以进行温和的三角化。同时通过100倍到200倍的放大率提取和弹出动脉段分离平滑肌肉细胞，同时产生内皮管。如有必要，请小心使用细尖钳子，将分离的切龙体和内部弹性层从内皮管中分离。确认所有平滑的肌肉细胞都分离，并且只有内皮细胞保持为完整的管。使用微操纵器，使用硅酸盐玻璃固定移液器将内皮管的每一端固定在超级熔管的玻璃盖滑道上。从腔室中洗掉分离的机度和平滑的肌肉细胞。用两个摩尔多的氯化钙PSS代替分离溶液。将移动平台与安全内皮管转移到超输液和实验钻机的显微镜上。然后在实验期间使用六个清洁的50毫升储液罐连续输送PSS和各自的药物溶液。使用直通流量控制阀手动将流量设置为与层流一致，同时将流量馈送与真空吸力相匹配。在记录背景数据和染料加载之前，将 PSS 送到腔室进行内皮管的超融合至少五分钟。要同时测量膜电位与细胞内钙浓度，拉一个尖锐的电极，并回填两个摩尔氯化钾，以记录从细胞加载毛皮-2染料。为了研究通过染料转移进行细胞内耦合，用溶解在两个摩尔氯化钾中的0.1%碘化基回填微电子。接下来，将电极放在用微操纵器固定在电表头台的移液器支架上，将电极放在涂有氯化物的银丝上。使用微操纵器将电极尖端短暂地定位到腔室中流动的 PSS 中，同时通过四倍目标查看。随后，将静膜电位设置为零，与接地浴电位一致。如果需要，请使用与电表相连的可听见的基线监视器将声高与潜在录音关联。之后，使用 40 倍的镜目标将放大倍数提高至 400 倍，并将电极尖端放在内皮管的电池上。使用光度测量软件调整光度学窗口，以专注于大约 50 到 80 个内皮细胞。然后使用微操纵器轻轻地将电极放入内皮管的一个单元中，等待至少两分钟，使静膜电位稳定下来。一旦静膜电位稳定在预期值，在没有光线的情况下打开荧光界面上的光电倍增管，开始通过刺激的 Fura-2 交替在 340 和 380 纳米，同时收集 510 纳米的荧光发射，获得细胞内钙浓度。一旦建立膜电位和细胞内钙浓度的同步测量，在药物应用之前，以恒定的层流速率，让内皮管以恒定的层流速率进行超输液约5分钟。将 PSS 中准备的药物以恒定的流速应用于超级输液室。在药物治疗期间，同时测量F340/F380比率中的膜电位。实验完成后，将电极从电池中取走。然后停止膜电位和细胞内钙浓度的分别记录，并保存文件进行数据分析。这是小鼠脑动脉内皮管的差分干扰对比图像，使用40倍的测量窗口进行实验调整。将锋利的电极放入电池中，如图像顶部所示。以下是用于同时测量 F340/F380 比率和膜电位以响应 100 微摩尔 ATP 的原始痕迹示例。按照这个程序，其他方法，如共和荧光显微镜，可以执行，以回答有关微域信号的内皮钙，和活性氧物种的其他问题，例如。一般来说，这项技术将继续为细胞生理学的研究铺平道路，以探索血液和淋巴循环中的血管功能和衰老。

Research

JoVE Journal

Biology

Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

近质膜和全球星形胶质细胞内钙动力学测量

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, ...

科研

生物学

Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents

Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents

CA同时测量人的心房肌细胞分离<sup&gt; +</sup&gt;瞬变和膜电流

The study of  electrophysiological properties of cardiac ion channels with the patch-clamp  technique and the exploration of cardiac cellular Ca2+ ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

活体显微镜成像的亚细胞结构的活体小鼠表达荧光蛋白

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

钙火花的评估在正常肌肉纤维

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

在肾小管上皮细胞胞浆钙的测量流式细胞仪

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

Single-channel Analysis and Calcium Imaging in the Podocytes of the Freshly Isolated Glomeruli

单通道分析和钙成像在新鲜分离的肾小球足细胞

Podocytes (renal glomerular epithelial cells) are known to regulate glomerular permeability and maintain glomerular structure; a key role for these cells ...

The Mouse Isolated Perfused Kidney Technique

鼠标离体肾技术

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

线粒体钙和线粒体膜的同时测量通过荧光显微镜活细胞的潜在

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ ...

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

分离高质量鼠心房和心室肌细胞，同时测量 Ca2+ 瞬变和 L 型钙电流

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...

Isolating and Imaging Live, Intact Pacemaker Regions of Mouse Renal Pelvis by Vibratome Sectioning

隔离和成像现场， 通过颤音分割老鼠肾佩尔维斯的精致起搏器区域

The renal pelvis (RP) is a funnel-shaped, smooth muscle structure that facilitates normal urine transport from the kidney to the ureter by regular, ...