Name: confocal imaging of neuropeptide y-phluorin: a technique to visualize insulin granule exocytosis in intact murine and human islets
Uploaded: 2017-09-13T13:00:00
Description: Watch this Scientific Journal Video about Confocal Imaging of Neuropeptide Y-pHluorin: A Technique to Visualize Insulin Granule Exocytosis in Intact Murine and Human Islets at JoVE.com

作者感谢马西娅 Boulina 从还原铁成像核心设施帮助与显微镜。这项工作得到了 NIH 赠款 1K01DK111757-01 (JA)、F31668418 (MM)、R01 DK111538、R33 ES025673 和 R56 DK084321 (AC) 的支持。

迈阿密大学动物伦理委员会批准了所有的实验. 
 1. 完整的孤立人或小鼠胰岛的病毒感染 <ol> <li> 胰岛文化: 准备胰岛培养基: 干诺医学研究实验室 (CMRL) 1066、10% (v/v) FBS 和2毫米 l-谷氨酰胺。
	<ol> <li> 人类胰岛是从综合胰岛分布计划 (NIDDK, NIH) 获得的。抵达后, 转移胰岛 (约500小岛), 以35毫米 non-tissue 培养皿2毫升 CMRL 培养基37和 #176; C, 5%/95% CO 2 /O 2 , 在病毒感染...

<ol>
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J., Martinez-Sanchez, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+beta-cell+identity,+glucose+sensing+and+the+control+of+insulin+secretion.">Pancreatic beta-cell identity, glucose sensing and the control of insulin secretion.</a> Biochem J. 466 (2), 203-218 (2015).</li><li>Rorsman, P., Renstrom, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin+granule+dynamics+in+pancreatic+beta+cells.">Insulin granule dynamics in pancreatic beta cells.</a> Diabetologia. 46 (8), 1029-1045 (2003).</li><li>Takahashi, N., Kishimoto, T., Nemoto, T., Kadowaki, T., Kasai, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fusion+pore+dynamics+and+insulin+granule+exocytosis+in+the+pancreatic+islet.">Fusion pore dynamics and insulin granule exocytosis in the pancreatic islet.</a> Science. 297 (5585), 1349-1352 (2002).</li><li>Ramirez, D. M., Khvotchev, M., Trauterman, B., Kavalali, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vti1a+identifies+a+vesicle+pool+that+preferentially+recycles+at+rest+and+maintains+spontaneous+neurotransmission.">Vti1a identifies a vesicle pool that preferentially recycles at rest and maintains spontaneous neurotransmission.</a> Neuron. 73 (1), 121-134 (2012).</li><li>Michael, D. J., Xiong, W., Geng, X., Drain, P., Chow, R. 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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+beta+cells+synthesize+neuropeptide+Y+and+can+rapidly+release+peptide+co-transmitters.">Pancreatic beta cells synthesize neuropeptide Y and can rapidly release peptide co-transmitters.</a> PLoS One. 6 (4), e19478 (2011).</li><li>Tsuboi, T., Rutter, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+forms+of+&amp;#34;kiss-and-run&amp;#34;+exocytosis+revealed+by+evanescent+wave+microscopy.">Multiple forms of &amp;#34;kiss-and-run&amp;#34; exocytosis revealed by evanescent wave microscopy.</a> Curr Biol. 13 (7), 563-567 (2003).</li><li>Zhu, D., 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=Synaptotagmin+I+and+IX+function+redundantly+in+controlling+fusion+pore+of+large+dense+core+vesicles.">Synaptotagmin I and IX function redundantly in controlling fusion pore of large dense core vesicles.</a> Biochem Biophys Res Commun. 361 (4), 922-927 (2007).</li><li>Aoki, 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=Duration+of+fusion+pore+opening+and+the+amount+of+hormone+released+are+regulated+by+myosin+II+during+kiss-and-run+exocytosis.">Duration of fusion pore opening and the amount of hormone released are regulated by myosin II during kiss-and-run exocytosis.</a> Biochem J. 429 (3), 497-504 (2010).</li><li>Felmy, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+cargo+release+from+dense+core+granules+by+size+and+actin+network.">Modulation of cargo release from dense core granules by size and actin network.</a> Traffic. 8 (8), 983-997 (2007).</li><li>Miesenbock, G., De Angelis, D. A., Rothman, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+secretion+and+synaptic+transmission+with+pH-sensitive+green+fluorescent+proteins.">Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins.</a> Nature. 394 (6689), 192-195 (1998).</li><li>Gandasi, N. 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=Survey+of+Red+Fluorescence+Proteins+as+Markers+for+Secretory+Granule+Exocytosis.">Survey of Red Fluorescence Proteins as Markers for Secretory Granule Exocytosis.</a> PLoS One. 10 (6), e0127801 (2015).</li><li>Almaca, 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=Spatial+and+temporal+coordination+of+insulin+granule+exocytosis+in+intact+human+pancreatic+islets.">Spatial and temporal coordination of insulin granule exocytosis in intact human pancreatic islets.</a> Diabetologia. 58 (12), 2810-2818 (2015).</li><li>Do, O. H., Low, J. T., Thorn, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lepr(db)+mouse+model+of+type+2+diabetes:+pancreatic+islet+isolation+and+live-cell+2-photon+imaging+of+intact+islets.">Lepr(db) mouse model of type 2 diabetes: pancreatic islet isolation and live-cell 2-photon imaging of intact islets.</a> J Vis Exp. (99), e52632 (2015).</li><li>Hanna, S. T., 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=Kiss-and-run+exocytosis+and+fusion+pores+of+secretory+vesicles+in+human+beta-cells.">Kiss-and-run exocytosis and fusion pores of secretory vesicles in human beta-cells.</a> Pflugers Arch. 457 (6), 1343-1350 (2009).</li><li>Carter, J. D., Dula, S. B., Corbin, K. L., Wu, R., Nunemaker, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+guide+to+rodent+islet+isolation+and+assessment.">A practical guide to rodent islet isolation and assessment.</a> Biol Proced Online. 11, 3-31 (2009).</li><li>Weber, 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=Adenoviral+transfection+of+isolated+pancreatic+islets:+a+study+of+programmed+cell+death+(apoptosis)+and+islet+function.">Adenoviral transfection of isolated pancreatic islets: a study of programmed cell death (apoptosis) and islet function.</a> J Surg Res. 69 (1), 23-32 (1997).</li><li>Michael, D. 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=Fluorescent+cargo+proteins+in+pancreatic+beta-cells:+design+determines+secretion+kinetics+at+exocytosis.">Fluorescent cargo proteins in pancreatic beta-cells: design determines secretion kinetics at exocytosis.</a> Biophys J. 87 (6), L03-L05 (2004).</li><li>Serre-Beinier, V., 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=Cx36+makes+channels+coupling+human+pancreatic+beta-cells,+and+correlates+with+insulin+expression.">Cx36 makes channels coupling human pancreatic beta-cells, and correlates with insulin expression.</a> Hum Mol Genet. 18 (3), 428-439 (2009).</li><li>Rutter, G. A., Hodson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beta+cell+connectivity+in+pancreatic+islets:+a+type+2+diabetes+target?.">Beta cell connectivity in pancreatic islets: a type 2 diabetes target?.</a> Cell Mol Life Sci. 72 (3), 453-467 (2015).</li><li>Shen, Y., Rosendale, M., Campbell, R. E., Perrais, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=pHuji,+a+pH-sensitive+red+fluorescent+protein+for+imaging+of+exo-+and+endocytosis.">pHuji, a pH-sensitive red fluorescent protein for imaging of exo- and endocytosis.</a> J Cell Biol. 207 (3), 419-432 (2014).</li><li>Speier, 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=Noninvasive+in+vivo+imaging+of+pancreatic+islet+cell+biology.">Noninvasive in vivo imaging of pancreatic islet cell biology.</a> Nat Med. 14 (5), 574-578 (2008).</li><li>Low, J. 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这份手稿描述了一种技术, 可以用来可视化胞胰岛素颗粒的β细胞在完整的胰岛的共聚焦显微镜。它使用 NPY-pHluorin 作为荧光记者克隆成腺病毒, 以确保高转染效率。
虽然该方法在我们手中是高效的, 但它可能需要一些修改, 主要取决于两个参数: 1) 胰岛的质量和 2) 的病毒库存的效价优化感染条件。在胰岛病毒感染之前, 允许胰岛准备在37° c 过夜时从隔离和运输压力中恢复。检?...

胰岛素是由胰岛的β细胞产生的, 它是葡萄糖代谢的关键调节器1。死亡或功能障碍的β细胞扰乱葡萄糖稳态和导致糖尿病2。胰岛素是包装在密集核心颗粒, 释放在一个 Ca2相关的方式3。阐明胰岛素颗粒胞是如何调节的, 是充分了解什么决定胰岛素分泌和开辟新的途径, 以确定治疗糖尿病的新型治疗靶点的关键。
胰岛素胞已被广泛研究使用电生理学方法, 如膜电容测量, 和显微方法结合荧光分子。膜电容测量具有良好的时间分辨率和允许单细胞记录。然而, 电容的变化反映了细胞的净表面变化, 不捕获单个融合事件或区分胰岛素颗粒融合与其他胰岛素分泌泡3。显微方法, 如双光子或全内反射荧光 (TIRF) 显微镜结合荧光探针和囊泡货物蛋白, 提供了额外的细节。这些技术捕获单个 exocytotic 事件以及前 post-exocytotic 阶段, 并可用于研究3单元人口中的 exocytotic 模式。
荧光记者可分为三种类型: 1) 胞外, 2) 细胞质, 或 3) 水泡。1) 细胞外的记者是极地示踪剂 (如, 糖, 磺 B (局), 路西法黄, pyranine), 可以通过细胞外环境介绍4。使用极性示踪剂可以对细胞中的融合孔进行调查, 并捕获各种胞间结构, 如血管。然而, 他们不报告囊泡货物行为。2) 细胞质记者是荧光探针耦合到膜相关蛋白, 面对细胞质和参与对接和胞。例子包括可溶性的N-ethylmaleimide 敏感因子附着蛋白受体 (网罗) 家族, 已成功地用于研究神经递质释放5神经科学。这种蛋白质有多个结合的伙伴, 而不是胰岛素颗粒的具体。3) 水泡记者是荧光探针融合到水泡货物蛋白质, 允许调查货物特定的泡状行为。胰岛素颗粒特定的货物蛋白包括胰岛素, c 肽, 胰岛淀粉样多肽, 和 NPY 等6,7。NPY 只是存在于胰岛素含有颗粒, 并与胰岛素 co-released, 使它成为一个优秀的合作伙伴的荧光记者8。
以前使用不同的荧光蛋白与 NPY 的融合来研究神经内分泌细胞中胞的各个方面, 如特定的结合异构体的要求9,10以及时效的释放取决于肌动蛋白骨架和肌球蛋白 II11,12。在这项研究中, 我们选择了 pHluorin 作为荧光记者, 这是一个修饰 GFP, 是荧光在酸性 ph 内致密核心颗粒, 但成为明亮的荧光后, 暴露于中性细胞外 ph 值13。成熟的胰岛素颗粒的酸性 pH 值低于5.5。一旦颗粒与等离子体膜融合并打开, 其货物就暴露在中性胞外 ph 值为 7.4, 允许使用 ph 敏感蛋白 pHluorin 作为记者7,14。
鉴于 pHluorin 的 pH 敏感性和胰岛素颗粒中 npy 的选择性表达, npy-pHluorin 融合结构可用于研究胰岛素颗粒胞的各种性质。病毒传递的融合结构确保高转染效率和工作在初级β细胞或细胞系, 以及在孤立的小岛。这种方法也可以作为研究胞在任何其他细胞类型与 NPY 包含囊泡的指导。它也可以与任何转基因小鼠模型结合, 研究某些条件 (knockdowns, 过度表达,等) 对胞的影响。这项技术以前被用来描述的空间和时间模式的胰岛素颗粒分泌的β细胞在人胰岛的人口15。

<table width="845">
	<tbody>
		<tr height="63">
			<td height="63" style="width:280px;height:63px;">Upright laser-scanning confocal microscope</td>
			<td style="width:232px;">Leica Microsystems, Wetzlar, Germany</td>
			<td style="width:167px;">TCS-SP5</td>
			<td style="width:167px;">includes LAS AF, the image acquisition software</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">Imaging chamber</td>
			<td style="width:232px;">Warner instruments</td>
			<td style="width:167px;">RC-26</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">Imaging chamber platform</td>
			<td style="width:232px;">Warner instruments</td>
			<td style="width:167px;">PH-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">22x40 glass coverslips</td>
			<td style="width:232px;">Daiggerbrand</td>
			<td style="width:167px;">G15972H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">Vacuum silicone grease</td>
			<td style="width:232px;">Sigma</td>
			<td style="width:167px;">Z273554-1EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">Multichannel perfusion system</td>
			<td style="width:232px;">Warner instruments</td>
			<td style="width:167px;">VC-8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">Single inline solution heater</td>
			<td style="width:232px;">Warner instruments</td>
			<td style="width:167px;">SH-27B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">Temperature controller</td>
			<td style="width:232px;">Warner instruments</td>
			<td style="width:167px;">TC-324C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">Peristaltic Suction pump</td>
			<td style="width:232px;">Pharmacia</td>
			<td style="width:167px;">P-1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:280px;height:42px;">35 mm Petri dish, non-tissue culture treated</td>
			<td style="width:232px;">VWR</td>
			<td style="width:167px;">10861-586</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">CMRL Medium, no glutamine</td>
			<td style="width:232px;">ThermoFisher</td>
			<td style="width:167px;">11530037</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">FBS, heat inactivated</td>
			<td style="width:232px;">ThermoFisher</td>
			<td style="width:167px;">16140071</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">L-Glutamine 200mM</td>
			<td style="width:232px;">ThermoFisher</td>
			<td style="width:167px;">25030081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">5M NaCl solution</td>
			<td style="width:232px;">Sigma</td>
			<td style="width:167px;">S5150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">3M KCl solution</td>
			<td style="width:232px;">Sigma</td>
			<td style="width:167px;">60135</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="width:280px;height:25px;">1M CaCl2 solution</td>
			<td style="width:232px;">Sigma</td>
			<td style="width:167px;">21115</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="width:280px;height:25px;">1M MgCl2 solution</td>
			<td style="width:232px;">Sigma</td>
			<td style="width:167px;">M1028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">Bovine Serum Albumin</td>
			<td style="width:232px;">Sigma</td>
			<td style="width:167px;">A2153</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">1M HEPES solution</td>
			<td style="width:232px;">Sigma</td>
			<td style="width:167px;">H0887</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">Vacuum filter</td>
			<td style="width:232px;">VWR</td>
			<td style="width:167px;">431098</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">D-Glucose</td>
			<td style="width:232px;">Sigma</td>
			<td style="width:167px;">G8270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">Poly-D-lysine hydrobromide</td>
			<td style="width:232px;">Sigma-aldrich</td>
			<td style="width:167px;">P6407</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">Di-8-ANNEP</td>
			<td style="width:232px;">ThermoFisher</td>
			<td style="width:167px;">D3167</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">3-isobutyl-1-methylxanthine (IBMX)</td>
			<td style="width:232px;">Sigma</td>
			<td style="width:167px;">I5879</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">Forskolin</td>
			<td style="width:232px;">Sigma</td>
			<td style="width:167px;">F3917</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

confocal imaging of neuropeptide y-phluorin: a technique to visualize insulin granule exocytosis in intact murine and human islets

胰岛素分泌在正常的生理条件下和疾病中起着核心作用。目前研究胰岛素颗粒胞的方法是使用电生理学或显微镜结合荧光记者的表达。然而, 大多数这些技术已被优化的克隆细胞系或需要游离胰岛。相比之下, 这里提出的方法可以实时显示胰岛素颗粒胞在完整的胰岛。在本议定书中, 我们首先描述的病毒感染的孤立胰岛与腺病毒, 编码 pH 敏感的绿色荧光蛋白 (GFP), pHluorin, 耦合到神经肽 Y (NPY)。其次, 我们描述了病毒感染后五天胰岛的共聚焦成像, 以及如何监测胰岛素颗粒的分泌。简要地, 被感染的小岛被放置在一个片在一个成像室和图像下的直立激光扫描共聚焦显微镜, 而不断灌注的胞外溶液含有各种刺激。共聚焦图像跨越50µm 的胰岛是获得作为延时录音使用的快速共振扫描仪。胰岛素颗粒与等离子体膜的融合可以随着时间的推移而随之而来。这个过程也允许测试一个电池的刺激在一个单一的实验, 是兼容的鼠标和人类的胰岛, 并可以结合各种染料的功能成像 (如, 膜电位或胞浆钙染料)。

该技术的整个工作流显示在图 1中。简单地, 老鼠或人的小岛可以感染腺病毒编码 NPY pHluorin 和影像, 在几天的文化, 在共聚焦显微镜下。当颗粒与等离子体膜和开放的保险丝, 荧光的增加观察和可以量化 (图 1)。为了确定 NPY-pHluorin 是否确实是一个合适的工具, 以监测胰岛素颗粒动力学, 感染的胰岛 immunostained 与抗体抗 GFP 和不同胰...

Watch this Scientific Journal Video about Confocal Imaging of Neuropeptide Y-pHluorin: A Technique to Visualize Insulin Granule Exocytosis in Intact Murine and Human Islets at JoVE.com

Confocal Imaging of Neuropeptide Y-pHluorin: A Technique to Visualize Insulin Granule Exocytosis in Intact Murine and Human Islets

我们描述了一个在完整的胰岛使用 pHluorin, pH 敏感的绿色荧光蛋白可视化的胞的协议。孤立的胰岛感染腺病毒编码 pHluorin 耦合到囊泡货物神经肽 Y。这使得检测胰岛素颗粒融合事件的共聚焦显微镜。

神经肽 Y-pHluorin 的共焦成像: 一种完整的小鼠和人胰岛胰岛素颗粒胞的可视化技术

Insulin secretion plays a central role in glucose homeostasis under normal physiological conditions as well as in disease. Current approaches to study ...

Research

JoVE Journal

Biology

Imaging Exocytosis in Retinal Bipolar Cells with TIRF Microscopy

TIRF显微镜视网膜双极细胞的影像胞外分泌

Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective ...

科研

生物学

Human Pancreatic Islet Isolation: Part II: Purification and Culture of Human Islets

人类胰岛的分离：第二部分：人类胰岛的分离纯化及文化

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of ...

A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells In situ

A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells In situ

一种技术，同时可视化病毒特异性CD8 + T细胞和病毒感染的细胞原位</em

The numbers and locations of virus-specific CD8+ T cells relative to the numbers and locations of their infected cell targets is thought to be critical in ...

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

注射胰岛素和血淋巴的提取来衡量成人胰岛素敏感性果蝇</em

Conserved nutrient sensing mechanisms exist between mammal and fruit fly where peptides resembling mammalian insulin and glucagon, respectively function ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

基于荧光测定活细胞中的钙进入商店经营：从培养的肿瘤细胞，骨骼肌纤维

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

单线粒体超氧闪烁在完整心脏或共聚焦成像<em&gt;在体内</em

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

高效的生产和纯化重组小鼠Kindlin-3从昆虫细胞生物物理研究

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

高分辨率成像定时，并在活体人脐静脉内皮细胞微管动力学自动分析

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

葡萄糖摄取测量和对胰岛素刺激的反应<em&gt;体外</em&gt;培养的人类初级肌管

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle ...