Name: culture of brain capillary pericytes for cytosolic calcium measurements and calcium imaging studies
Uploaded: 2020-05-27T15:00:00
Description: Watch this Scientific Journal Video about Culture of Brain Capillary Pericytes for Cytosolic Calcium Measurements and Calcium Imaging Studies at JoVE.com

作者希望感谢伦德贝克基金会关于脑障碍和药物输送的研究倡议（RIBBDD）和西蒙·胡格纳斯家庭基金会的资助。

1. 为细胞培养准备缓冲液和溶液
<ol><li>在4°C下，在50 mL的PBS中溶解5毫克胶原蛋白IV，准备胶原蛋白库存溶液。 将库存溶液分成5 mL部分，储存在-20°C。</li>
	<li>在5mL的无菌水中溶解5毫克纤维素，准备纤维素库存溶液。将纤维素储存在 -20 °C 下以 500 μL 的等分。 解冻时，将 PBS 添加到 50 mL 的最终体积中，以准备工作解决方案并将其储存在 4°C。</li>
	<li>准备Dulbecco的改性鹰介质（DMEM）完整的介质，?...

<ol>
	<li>Armulik, A., Genove, G., Betsholtz, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pericytes:+Developmental,+Physiological,+and+Pathological+Perspectives,+Problems,+and+Promises.">Pericytes: Developmental, Physiological, and Pathological Perspectives, Problems, and Promises.</a> Developmental Cell. 21 (2), 193-215 (2011).</li><li>Abbott, N. J., Ronnback, L., Hansson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte-endothelial+interactions+at+the+blood-brain+barrier.">Astrocyte-endothelial interactions at the blood-brain barrier.</a> Nature Reviews Neuroscience. 7 (1), 41-53 (2006).</li><li>Mathiisen, T. M., Lehre, K. P., Danbolt, N. C., Ottersen, O. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Perivascular+Astroglial+Sheath+Provides+a+Complete+Covering+of+the+Brain+Microvessels:+An+Electron+Microscopic+3D+Reconstruction.">The Perivascular Astroglial Sheath Provides a Complete Covering of the Brain Microvessels: An Electron Microscopic 3D Reconstruction.</a> Glia. 58 (9), 1094-1103 (2010).</li><li>Helms, H. C., 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=In+vitro+models+of+the+blood-brain+barrier:+An+overview+of+commonly+used+brain+endothelial+cell+culture+models+and+guidelines+for+their+use.">In vitro models of the blood-brain barrier: An overview of commonly used brain endothelial cell culture models and guidelines for their use.</a> Journal of Cerebral Blood Flow and Metabolism. 36 (5), 862-890 (2016).</li><li>Zhou, L., Sohet, F., Daneman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+Pericytes+from+Rodent+Optic+Nerve+by+Immunopanning.">Purification of Pericytes from Rodent Optic Nerve by Immunopanning.</a> Cold Spring Harbor Protocols. , 608-617 (2014).</li><li>Liu, G. 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=Isolation,+Purification,+and+Cultivation+of+Primary+Retinal+Microvascular+Pericytes:+A+Novel+Model+Using+Rats.">Isolation, Purification, and Cultivation of Primary Retinal Microvascular Pericytes: A Novel Model Using Rats.</a> Microcirculation. 21 (6), 478-489 (2014).</li><li>Nayak, R. C., Herman, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bovine+retinal+microvascular+pericytes:+isolation,+propagation,+and+identification.">Bovine retinal microvascular pericytes: isolation, propagation, and identification.</a> Methods In Molecular Medicine. 46, 247-263 (2001).</li><li>Nees, S., 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,+bulk+cultivation,+and+characterization+of+coronary+microvascular+pericytes:+the+second+most+frequent+myocardial+cell+type+in+vitro.">Isolation, bulk cultivation, and characterization of coronary microvascular pericytes: the second most frequent myocardial cell type in vitro.</a> American Journal of Physiology-Heart and Circulatory Physiology. 302 (1), H69-H84 (2012).</li><li>Armulik, A., Abramsson, A., Betsholtz, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial/pericyte+interactions.">Endothelial/pericyte interactions.</a> Circulation Research. 97 (6), 512-523 (2005).</li><li>Helms, H. C., Brodin, B., Milner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+in+Molecular+Biology.">Methods in Molecular Biology.</a> Cerebral Angiogenesis: Methods and Protocols. 1135, 365-382 (2014).</li><li>Abbracchio, M. P., Burnstock, G., Verkhratsky, A., Zimmermann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purinergic+signalling+in+the+nervous+system:+an+overview.">Purinergic signalling in the nervous system: an overview.</a> Trends in Neurosciences. 32 (1), 19-29 (2009).</li><li>Cai, C., 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=Stimulation-induced+rises+in+cerebral+blood+flow+and+local+capillary+vasoconstriction+depend+on+conducted+vascular+responses+in+brain+capillaries.">Stimulation-induced rises in cerebral blood flow and local capillary vasoconstriction depend on conducted vascular responses in brain capillaries.</a> PNAS. , (2018).</li><li>Tigges, U., Welser-Alves, J. V., Boroujerdi, A., Milner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+and+simple+method+for+culturing+pericytes+from+mouse+brain.">A novel and simple method for culturing pericytes from mouse brain.</a> Microvascular Research. 84 (1), 74-80 (2012).</li><li>Maier, C. L., Shepherd, B. R., Yi, T., Pober, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Explant+Outgrowth,+Propagation+and+Characterization+of+Human+Pericytes.">Explant Outgrowth, Propagation and Characterization of Human Pericytes.</a> Microcirculation. 17 (5), 367-380 (2010).</li><li>Orlidge, A., Damore, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+specific+effects+of+glycosaminoglycans+on+the+attachment+and+proliferation+of+vascular+wall+components.">Cell specific effects of glycosaminoglycans on the attachment and proliferation of vascular wall components.</a> Microvascular Research. 31 (1), 41-53 (1986).</li><li>Rhinehart, K., Zhang, Z., Pallone, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca2++signaling+and+membrane+potential+in+descending+vasa+recta+pericytes+and+endothelia.">Ca2+ signaling and membrane potential in descending vasa recta pericytes and endothelia.</a> American Journal of Physiology-Renal Physiology. 283, F852-F860 (2002).</li><li>Bintig, W., 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=Purine+receptors+and+Ca2++signalling+in+the+human+blood-brain+barrier+endothelial+cell+line+hCMEC/D3.">Purine receptors and Ca2+ signalling in the human blood-brain barrier endothelial cell line hCMEC/D3.</a> Purinergic Signalling. 8 (1), 71-80 (2012).</li><li>Neuhaus, A. A., Couch, Y., Sutherland, B. A., Buchan, 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=Novel+method+to+study+pericyte+contractility+and+responses+to+ischaemia+in+vitro+using+electrical+impedance.">Novel method to study pericyte contractility and responses to ischaemia in vitro using electrical impedance.</a> Journal of Cerebral Blood Flow and Metabolism. 37 (6), 2013-2024 (2017).</li><li>Kamouchi, 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=Calcium+influx+pathways+in+rat+CNS+pericytes.">Calcium influx pathways in rat CNS pericytes.</a> Molecular Brain Research. 126 (2), 114-120 (2004).</li><li>Das, 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=ATP+Causes+Retinal+Pericytes+to+Contract+In+vitro.">ATP Causes Retinal Pericytes to Contract In vitro.</a> Experimental Eye Research. 46, 349-362 (1988).</li></ol>

在这项研究中，我们提出了一种将原发性胸椎动物与牛脑分离的方法。所述协议允许培养这种本来相当不可接近的单元类型。随后获得的细胞培养物是近同质的性年期，以细胞形态和蛋白质表达12为基础，内皮细胞和胶质细胞几乎没有污染。此外，我们演示了一种简单明了的方法，使用两种不同的方法，根据预期结果，用钙染料为 Ca2+成像加载过白剂。
<p class="jove_conte...

脑毛细管内皮，连同内皮细胞和星形细胞，构成NVU1，2，3。内皮细胞构成毛细血管的结构基础，形成直径为5-8μm的长圆柱管。内皮细胞零星被围皮覆盖，周围是星形细胞的突起;星细胞末脚。
血脑屏障（BBB）位于脑毛细血管，是大脑和血液之间交换营养物质、气体和废物的主要地点。BBB还保护大脑免受内源和外源性神经毒素的污染，并充当大量药物化合物的传递屏障。屏障功能是制药公司开发中枢神经系统（CNS）药物的重点领域，也是障碍。这激起了对调查NVU在培养中细胞的很大兴趣。脑星形细胞和内皮细胞在一些研究中被培养和描述，而对周来细胞培养的研究和协议是稀疏的。
此前公布的协议在一定程度上描述了脑毛细管的生成，使用一系列不同的方法，如免疫平移5、高和低血糖介质6、荧光激活细胞分选7、密度梯度离心8等。虽然这些方法似乎足以获得过长的区域性，但有些是耗时的，成本昂贵，获得可能不理想，因为文化通道的数量，可以去区分的周分。此外，培养的周利未得到体外信号研究的潜力，直到现在还相当未被探索。
本工作的重点是从孤立的牛脑毛细血管中生成腹膜培养物，以及随后对细胞内钙（一个重要的细胞内第二信使）变化的测量和成像研究进行的后续设置。我们简要地描述了毛细血管与皮质灰质的分离（详情见Helms等人10），以及纯单培养中胸腺的分离和培养，而不受到内皮或胶质细胞的污染。然后，我们提供一个协议，用于在96孔板中播种性，并为钙探针Fura-2 AM提供加载协议。最后，我们介绍了在显微镜培养室中如何将过色物用于实时共合成像，并描述了其方案。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ATP</td>
			<td>Tocris</td>
			<td>3245</td>
			<td></td>
		</tr>
		<tr>
			<td>Cal-520 AM</td>
			<td>AAT Bioquest</td>
			<td>21130</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell incubator</td>
			<td>Thermo Fisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Thermo Fisher</td>
			<td>Heraeus Multifuge 3SR+</td>
			<td>Standard large volume centrifuge for spinning down cells</td>
		</tr>
		<tr>
			<td>Collagen IV</td>
			<td>Sigma Aldrich</td>
			<td>C5533</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal laser scanning microscope</td>
			<td>Carl Zeiss</td>
			<td>Zeiss LSM 510</td>
			<td>Inverted microscope</td>
		</tr>
		<tr>
			<td>Counting chamber</td>
			<td>FastRead</td>
			<td>102</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslip cell chamber</td>
			<td>Airekacells</td>
			<td>SC15022</td>
			<td></td>
		</tr>
		<tr>
			<td>Cremophor EL</td>
			<td>Sigma Aldrich</td>
			<td>C5135</td>
			<td>Formerly known as Kolliphor EL</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma Aldrich</td>
			<td>471267</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagles Medium</td>
			<td>Sigma Aldrich</td>
			<td>D0819</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>PAA/GE Healthcare</td>
			<td>A15-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Sigma Aldrich</td>
			<td>F1141</td>
			<td></td>
		</tr>
		<tr>
			<td>Fura-2 AM</td>
			<td>Thermo Fisher</td>
			<td>F1201</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslips 22x22 mm</td>
			<td>VWR International</td>
			<td>631-0123</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Gibco</td>
			<td>14065-049</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma Aldrich</td>
			<td>H3149</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>AppliChem Panreac</td>
			<td>A1069</td>
			<td></td>
		</tr>
		<tr>
			<td>Light microscope</td>
			<td>Olympus</td>
			<td>Olympus CK2</td>
			<td>Upright light microscope with phase contrast</td>
		</tr>
		<tr>
			<td>MEM nonessential amino acids</td>
			<td>Sigma Aldrich</td>
			<td>M7145</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate Reader</td>
			<td>BMG LabTech</td>
			<td>NOVOstar</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma Aldrich</td>
			<td>D8537</td>
			<td>Phosphate-buffered saline</td>
		</tr>
		<tr>
			<td>penicillin G sodium/streptomycin sulfate</td>
			<td>Sigma Aldrich</td>
			<td>P0781</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic F127</td>
			<td>Sigma Aldrich</td>
			<td>P2443</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Sigma Aldrich</td>
			<td>T4299</td>
			<td></td>
		</tr>
		<tr>
			<td>T-75 flask</td>
			<td>Sigma Aldrich</td>
			<td>CLS3972</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>Corning incorporated</td>
			<td>3603</td>
			<td></td>
		</tr>
	</tbody>
</table>

culture of brain capillary pericytes for cytosolic calcium measurements and calcium imaging studies

在称为神经血管单元（NVU）的结构中，佩里西特与内皮细胞和星形末脚相关。脑毛细管性周丽素功能不完全知道。已建议参与毛细管发育、内皮屏障紧绷和转膜活动调节、毛细管音的调节，并在某些大脑病理中发挥关键作用。
由于在脑筋障碍中难以可视化过程，以及接近 NVU 的其他细胞，因此在完整大脑中对研究是具有挑战性的。本协议描述了一种主要牛脑毛细管性胸毛的分离和培养方法及其在钙成像研究中的以下用法，其中可以研究参与大脑信号和病理学的激动剂的影响。皮质毛细管碎片可以附着在培养瓶的底部，6天后，内皮细胞和心状物从毛细管碎片中生长出来。内皮细胞通过温和的三分血化去除，在通过前再培养5天。
分离的腹膜在96孔培养板中播种，并装有钙指示器染料（Fura-2醋氧甲基（AM），用于测量板读卡器设置中的细胞内钙水平。或者，在盖玻片上播种，并安装在细胞室中。在用钙指示器（Cal-520 AM）加载后，可以使用共物显微镜在488纳米和发射波长为510-520 nm时进行钙活成像。
此处描述的方法已用于从原发性脑毛细管腹膜获得第一个细胞内钙测量，表明环状细胞通过 ATP 受到刺激，并且能够在体外收缩。

牛脑毛细管从新鲜脑组织分离 出来，图1 呈现了毛细管播种和细胞生长在几天内，随后的胸腺细胞纯化。毛细管在第1天完全附着在烧瓶上，第2天内皮发芽变得可见（图1，第2天）。4天后，细胞外生长高度独特（图1，第4a天），内皮细胞根据所述协议通过温和的三辛化被去除。毛细管的残余物在试鸣后可?...

Watch this Scientific Journal Video about Culture of Brain Capillary Pericytes for Cytosolic Calcium Measurements and Calcium Imaging Studies at JoVE.com

Culture of Brain Capillary Pericytes for Cytosolic Calcium Measurements and Calcium Imaging Studies

脑毛细管性信号是调节血脑屏障特性和血流的基本参与者。该协议描述了如何分离、培养脑毛细管腹膜，在细胞类型方面进行特征分析，并应用于用荧光探针研究细胞内钙信号。

脑毛细管中培养细胞细胞钙测量和钙成像研究

Pericytes are associated with endothelial cells and astrocytic endfeet in a structure known as the neurovascular unit (NVU). Brain capillary pericyte ...

这是一种简单而有效的方法来隔离和培养原脑环状物，用于研究细胞间钙信号。获得的细胞培养物是近同质的中白细胞种群，加载膜状物进行钙成像非常简单。这里描述的方法应该为该领域的其他研究人员提供强大的工具，用于研究腹膜生物学，以及体外腹膜内信号。首先在37摄氏度的水浴中解冻一小瓶毛细管。毛细管解冻后，将它们转移到具有 30 毫升 DMEM 复合剂的离心管中，并在 500 倍 G 下将其离心 5 分钟，然后从管中取出介质，并在 10 毫升新鲜 DMEM comp 中重新悬挂调色板。将悬浮液转移到涂有涂层的 T-75 烧瓶中，让毛细管在 37 摄氏度的培养箱中粘附在底部 4 到 6 个小时，孵化器提供 10% 的二氧化碳。孵育后，在光学显微镜下检查烧瓶。毛细管的分馏现在应该附在烧瓶的底部。在播种毛细管后的第四天，在显微镜下检查它们，以确保它们大约为 60 到 70% 的汇合。吸气介质，用PBS轻轻清洗细胞。加入两毫升解冻的三辛-EDTA，将烧瓶留在培养箱中一至三分钟，经常将烧瓶中取出，用显微镜观察细胞分离。当内皮细胞开始四舍五入时，轻轻敲击烧瓶将其分离。当细胞分离时，通过向烧瓶中添加 10 毫升 DMEM 复合剂来停止尝试性。用介质冲洗烧瓶几次，并吸入内皮细胞悬浮液，现在可用于其他目的。在烧瓶中加入10毫升DMEM复合物，并在光学显微镜下检查，以确保心状物仍然存在并附着在底部。然后，将烧瓶放回孵化器，让富含字节的培养者生长。要将膜覆盖的 96 孔板中播种，请将它们分离，如文本手稿中所述。吸气介质，注意不要干扰细胞颗粒，并在一毫升新鲜DMEM复合中重新悬浮颗粒。使用计数室计算细胞，然后计算每井应添加到每个井的悬浮量，以每井播种 10，000 个细胞。添加细胞悬浮液，然后向细胞添加足够的DMEM复合，以达到每孔200微升的总体积。当准备用 Fura-2 AM 钙指示器染料加载心状物时，将 96 孔板与细胞一起从培养箱中取出，然后从孔中吸出介质。用测定缓冲液清洗细胞两次，并在每井中加入100微升的装载溶液。用锡箔包裹盘子，以避免照片漂白，并在室温下孵育45分钟，30 RPM摇动。孵育后，吸气缓冲液，用测定缓冲液洗涤细胞两次。将100微升新鲜剂加入检测缓冲液，让细胞孵育30分钟。孵育后，将板读卡器的温度设置为37摄氏度，将带细胞的96井板转移到样品板位置，然后将带激动剂的试剂板放在试剂板位置。首先测量电池的负载，以确保 Fura-2 AM 在所有孔中负载相等，然后在 340 至 380 纳米时以激发荧光波长进行测量，在 510 纳米时执行发射波长。加入50微升的激动剂，以150微升/秒的速度，从试剂盘到每一个带细胞的井。将盖板插入电池室，然后拧紧，以避免泄漏。将 DMEM 复合和计算的细胞悬浮量添加到每个腔室中，最终体积为 500 微升。在37摄氏度和10%的二氧化碳下孵育细胞室6天，或直到汇合。一旦细胞汇合，将细胞室从培养箱中取出，并吸气培养。用测定缓冲液清洗细胞两次，在每个腔室中加入500微升的加载缓冲液，然后在室温下孵育45分钟。孵育后，吸气缓冲液，用检测缓冲液清洗细胞两次，然后在每个腔室中加入500微升新鲜检测缓冲液，并在室温下再孵育30分钟，使 AM 酯的裂解。用 500 微升新鲜测定缓冲液更换缓冲液，并在共和显微镜下进行实时成像。尽可能轻轻地将细胞室安装在显微镜的舞台上，以避免细胞受到干扰。将激励波长设置为 488 纳米，发射为 515 纳米，以 5 秒间隔进行 2 分钟顺序图像采集，将 x，y 图像大小设置为 512 x 512 像素。向细胞室添加 300 微升 100 毫摩尔 ATP，然后启动图像采集。该协议用于净化牛脑毛细血管的胸毛。在九天中，从种子毛细血管中生长的细胞生长被成像出来。毛细管在第一天完全附着在烧瓶上，第二天，内皮发芽是可见的。四天后，细胞外生长明显，内皮细胞通过三位一体机切除。毛细管的残余物在试鸣后存在，但几天后从烧瓶中消失。去除内皮层后，允许内皮层生长至汇合。第九天，周利群岛达到约80%的汇合，形成岛屿。将ATP添加到富拉-2加载的环状物导致细胞酸钙水平增加。反应发生在ATP加入过周后，并在测量的时间段缓慢下降。钙反应也测量与实时共体成像。测量了基线荧光，然后在64秒内加入ATP，细胞内钙反应强烈。不久之后，细胞酸钙在细胞中分体化，细胞面积减少可见。到300秒时，细胞面积大减，荧光几乎降低到基线水平。这种方法已被用于从原发性脑毛细管腹膜获得第一个细胞内钙测量，表明腹膜通过ATP刺激，并能够在体外收缩。

Research

JoVE Journal

Neuroscience

Functional Calcium Imaging in Developing Cortical Networks

在开发皮层网络功能钙成像

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in ...

科研

神经科学

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

钙成像的急性下区片的制备

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural ...

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

脑切片神经元树突的双光子钙成像

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

Ablation of a Neuronal Population Using a Two-photon Laser and Its Assessment Using Calcium Imaging and Behavioral Recording in Zebrafish Larvae

用双光子激光消融神经种群及其对斑马鱼幼虫钙成像和行为记录的评价

To identify the role of a subpopulation of neurons in behavior, it is essential to test the consequences of blocking its activity in living animals. Laser ...

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

前体钙成像用于可视化大脑对果蝇内分泌信号的反应

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model ...

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

活体转基因小鼠脑围产层钙和血液学成像

Recent advances in protein biology and mouse genetics have made it possible to measure intracellular calcium fluctuations of brain cells in vivo and to ...

Stereotaxic Viral Injection and Gradient-Index Lens Implantation for Deep Brain In Vivo Calcium Imaging

Stereotaxic Viral Injection and Gradient-Index Lens Implantation for Deep Brain In Vivo Calcium Imaging

A miniature fluorescence microscope (miniscope) is a potent tool for in vivo calcium imaging from freely behaving animals. It offers several advantages ...

Subcellular Imaging of Neuronal Calcium Handling In Vivo

Subcellular Imaging of Neuronal Calcium Handling In Vivo

体内神经元钙处理的亚细胞成像

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a ...

Two-Photon and Wide-Field Calcium Imaging in the Superior Colliculus

Calcium Imaging in Mouse Superior Colliculus

Unveiling Neural Coding and Mechanisms of Visual Processing in the Superior Colliculus (Video) | JoVE

小鼠上丘的钙成像

The superior colliculus (SC), an evolutionarily conserved midbrain structure in all vertebrates, is the most sophisticated visual center before the ...