Name: visualization of endogenous mitophagy complexes in situ in human pancreatic beta cells utilizing proximity ligation assay
Uploaded: 2019-05-02T14:00:00
Description: Watch this Scientific Journal Video about Visualization of Endogenous Mitophagy Complexes In Situ in Human Pancreatic Beta Cells Utilizing Proximity Ligation Assay at JoVE.com

作者感谢JDRF(CDA-2016-189和SRA-2018-539)、国家糖尿病、消化和肾脏疾病研究所、国家卫生研究院(R01-DK-108921)、布雷姆家族和安东尼家族的资助。JDRF职业发展奖由丹麦糖尿病学院获得部分支持,该学院由诺和诺德基金会支持。

使用已识别的捐赠者人类胰腺胰岛是通过机构审查委员会(IRB)豁免,并符合密歇根大学IRB政策。人类胰腺胰岛由NIH/NIDDK赞助的综合胰岛分布方案(IIDP)提供。
1. 人类小岛样品制备
<ol><li>
单细胞分离
	<ol>
<li>培养人类胰岛样品(4000~6000胰岛当量/10 mL介质),在胰腺胰岛介质(PIM(S))介质中至少1天,辅以1 mM谷氨酰胺(PIM(G))、100单位/mL抗霉菌抗生素、1mM丙酸钠和10%胎儿牛血清(FBS)?...

<ol>
	<li>Pearson, G., 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=Clec16a,+Nrdp1,+and+USP8+Form+a+Ubiquitin-Dependent+Tripartite+Complex+That+Regulates+beta-Cell+Mitophagy.">Clec16a, Nrdp1, and USP8 Form a Ubiquitin-Dependent Tripartite Complex That Regulates beta-Cell Mitophagy.</a> Diabetes. 67 (2), 265-277 (2018).</li><li>Soleimanpour, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+diabetes+susceptibility+gene+Clec16a+regulates+mitophagy.">The diabetes susceptibility gene Clec16a regulates mitophagy.</a> Cell. 157 (7), 1577-1590 (2014).</li><li>Kaufman, B. A., Li, C., Soleimanpour, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+regulation+of+&#946;-cell+function:+Maintaining+the+momentum+for+insulin+release.">Mitochondrial regulation of &#946;-cell function: Maintaining the momentum for insulin release.</a> Molecular Aspects of Medicine. , (2015).</li><li>Hattori, N., Saiki, S., Imai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+by+mitophagy.">Regulation by mitophagy.</a> The International Journal of Biochemistry &amp; Cell Biology. 53, 147-150 (2014).</li><li>Soleimanpour, S. 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=Diabetes+Susceptibility+Genes+Pdx1+and+Clec16a+Function+in+a+Pathway+Regulating+Mitophagy+in+beta-Cells.">Diabetes Susceptibility Genes Pdx1 and Clec16a Function in a Pathway Regulating Mitophagy in beta-Cells.</a> Diabetes. 64 (10), 3475-3484 (2015).</li><li>Zhong, L., Tan, Y., Zhou, A., Yu, Q., Zhou, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RING+Finger+Ubiquitin-Protein+Isopeptide+Ligase+Nrdp1/FLRF+Regulates+Parkin+Stability+and+Activity.">RING Finger Ubiquitin-Protein Isopeptide Ligase Nrdp1/FLRF Regulates Parkin Stability and Activity.</a> Journal of Biological Chemistry. 280 (10), 9425-9430 (2005).</li><li>Durcan, T. 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=USP8+regulates+mitophagy+by+removing+K6-linked+ubiquitin+conjugates+from+parkin.">USP8 regulates mitophagy by removing K6-linked ubiquitin conjugates from parkin.</a> The EMBO Journal. 33 (21), 2473-2491 (2014).</li><li>Gauthier, T., Claude-Taupin, A., Delage-Mourroux, R., Boyer-Guittaut, M., Hervouet, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proximity+Ligation+In+situ+Assay+is+a+Powerful+Tool+to+Monitor+Specific+ATG+Protein+Interactions+following+Autophagy+Induction.">Proximity Ligation In situ Assay is a Powerful Tool to Monitor Specific ATG Protein Interactions following Autophagy Induction.</a> PLoS ONE. 10 (6), e0128701 (2015).</li><li>Silva Xavier, D. a., G, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Cells+of+the+Islets+of+Langerhans.">The Cells of the Islets of Langerhans.</a> Journal of Clinical Medicine. 7 (3), 54 (2018).</li><li>Steiner, D. J., Kim, A., Miller, K., Hara, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+islet+plasticity:+Interspecies+comparison+of+islet+architecture+and+composition.">Pancreatic islet plasticity: Interspecies comparison of islet architecture and composition.</a> Islets. 2 (3), 135-145 (2010).</li><li>Cabrera, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+unique+cytoarchitecture+of+human+pancreatic+islets+has+implications+for+islet+cell+function.">The unique cytoarchitecture of human pancreatic islets has implications for islet cell function.</a> Proceedings of the National Academy of Sciences. 103 (7), 2334 (2006).</li><li>Brissova, 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=Assessment+of+Human+Pancreatic+Islet+Architecture+and+Composition+by+Laser+Scanning+Confocal+Microscopy.">Assessment of Human Pancreatic Islet Architecture and Composition by Laser Scanning Confocal Microscopy.</a> Journal of Histochemistry and Cytochemistry. 53 (9), 1087-1097 (2005).</li><li>Babu, D. A., Deering, T. G., Mirmira, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+feat+of+metabolic+proportions:+Pdx1+orchestrates+islet+development+and+function+in+the+maintenance+of+glucose+homeostasis.">A feat of metabolic proportions: Pdx1 orchestrates islet development and function in the maintenance of glucose homeostasis.</a> Molecular Genetics and Metabolism. 92 (1), 43-55 (2007).</li><li>Poitout, 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=Glucolipotoxicity+of+the+pancreatic+beta+cell.">Glucolipotoxicity of the pancreatic beta cell.</a> Biochimica Biophysica Acta. 1801 (3), 289-298 (2010).</li><li>Pearson, G., Soleimanpour, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+ubiquitin-dependent+mitophagy+complex+maintains+mitochondrial+function+and+insulin+secretion+in+beta+cells.">A ubiquitin-dependent mitophagy complex maintains mitochondrial function and insulin secretion in beta cells.</a> Autophagy. 14 (7), 1160-1161 (2018).</li><li>Li, 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=Single-cell+transcriptomes+reveal+characteristic+features+of+human+pancreatic+islet+cell+types.">Single-cell transcriptomes reveal characteristic features of human pancreatic islet cell types.</a> EMBO Reports. 17 (2), 178-187 (2016).</li><li>DiGruccio, M. 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=Comprehensive+alpha,+beta+and+delta+cell+transcriptomes+reveal+that+ghrelin+selectively+activates+delta+cells+and+promotes+somatostatin+release+from+pancreatic+islets.">Comprehensive alpha, beta and delta cell transcriptomes reveal that ghrelin selectively activates delta cells and promotes somatostatin release from pancreatic islets.</a> Molecular Metabolism. 5 (7), 449-458 (2016).</li><li>Lawlor, N., 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=Single-cell+transcriptomes+identify+human+islet+cell+signatures+and+reveal+cell-type&#8211;specific+expression+changes+in+type+2+diabetes.">Single-cell transcriptomes identify human islet cell signatures and reveal cell-type&#8211;specific expression changes in type 2 diabetes.</a> Genome Research. 27 (2), 208-222 (2017).</li><li>Dorrell, 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=Human+islets+contain+four+distinct+subtypes+of+&#946;+cells.">Human islets contain four distinct subtypes of &#946; cells.</a> Nature Communications. 7, 11756 (2016).</li><li>Dorrell, 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=Isolation+of+major+pancreatic+cell+types+and+long-term+culture-initiating+cells+using+novel+human+surface+markers.">Isolation of major pancreatic cell types and long-term culture-initiating cells using novel human surface markers.</a> Stem Cell Research. 1 (3), 183-194 (2008).</li><li>Jayaraman, 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+novel+method+for+the+detection+of+viable+human+pancreatic+beta+cells+by+flow+cytometry+using+fluorophores+that+selectively+detect+labile+zinc,+mitochondrial+membrane+potential+and+protein+thiols.">A novel method for the detection of viable human pancreatic beta cells by flow cytometry using fluorophores that selectively detect labile zinc, mitochondrial membrane potential and protein thiols.</a> Cytometry Part A. 73 (7), 615-625 (2008).</li><li>Arda, H. E., 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=Age-Dependent+Pancreatic+Gene+Regulation+Reveals+Mechanisms+Governing+Human+&#223;-Cell+Function.">Age-Dependent Pancreatic Gene Regulation Reveals Mechanisms Governing Human &#223;-Cell Function.</a> Cell Metabolism. 23 (5), 909-920 (2016).</li><li>Allen, G. F. G., Toth, R., James, J., Ganley, I. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+iron+triggers+PINK1/Parkin-independent+mitophagy.">Loss of iron triggers PINK1/Parkin-independent mitophagy.</a> EMBO Reports. 14 (12), 1127-1135 (2013).</li><li>Villa, E., Marchetti, S., Ricci, 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=No+Parkin+Zone:+Mitophagy+without+Parkin.">No Parkin Zone: Mitophagy without Parkin.</a> Trends in Cell Biology. , (2018).</li></ol>

在这里,我们描述了一种简单而有效的方法,用于在感兴趣的组织/细胞中使用NRDP1:USP8 PLA,以量化上游米托巴细胞复合物的形成。我们之前通过多种方法确认在胰腺β细胞中形成CLEC16A-NRDP1-USP8米托巴细胞复合物,包括共免疫沉淀实验、无细胞相互作用研究、体外和基于细胞泛化测定,并演示了这个复杂驱动调节米托巴哈通通1,15。我们在这里报告和描述的PLA研究...

胰腺β细胞产生维持正常葡萄糖平衡所需的胰岛素,其失败导致所有形式的糖尿病的发展。Β细胞保持强大的线粒体能力,以产生将葡萄糖代谢与胰岛素释放耦合所需的能量。最近,很明显,维持功能线粒体质量对于最佳β细胞功能1,2,3至关重要。为了维持功能线粒体质量,β细胞依靠质量控制机制来去除功能失调、受损或老化的线粒体4。我们和其他人以前已经证明,β细胞依靠一种特殊形式的线粒体周转,称为线粒体自噬(或线粒体),以保持线粒体质量控制在啮齿动物和人类胰岛1, 2,5.然而,不幸的是,在人类胰腺β细胞中,没有简单的方法来检测米托巴细胞或内分泌的米托巴细胞成分。
我们最近已经表明,β细胞中米托巴的上游调节依赖于形成一种蛋白质复合物,包括E3利气CLEC16A和NRDP1和二苯基酸酶USP81。NRDP1和USP8已经独立显示,通过行动对关键的米托巴沙第6,7影响米托巴吉。NRDP1的目标是PARKIN的泛化和降解关闭米托巴希6,和USP8专门二苯基丁基丁K6链接的PARKIN,以促进其易位线粒体7。接近结扎测定(PLA)技术是蛋白质相互作用生物学领域的一个最新进展,允许在单个细胞中原位实现内源性蛋白质相互作用的可视化,不受稀缺样品材料的限制。这种方法对人类小岛/β细胞生物学特别诱人,因为样品可用性的稀疏性,加上需要了解异质细胞类型中生理相关的蛋白质复合物。
利用PLA方法,我们能够观察原发性人类胰腺β细胞和神经元细胞系中的关键内源性米托巴细胞复合物,并演示糖尿病环境对米托巴病途径1的影响。总之,该协议的首要目标是分析缺乏丰富物质的组织中的特定米托巴状蛋白复合物,或者无法进行常规蛋白质相互作用研究。

<table>
	<tbody>
		<tr>
			<td>0.25% trypsin-EDTA 1X</td>
			<td>Life Technologies</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic</td>
			<td>Life Technologies</td>
			<td>15240-062</td>
			<td></td>
		</tr>
		<tr>
			<td>Block solution</td>
			<td>Homemade</td>
			<td></td>
			<td>Use 1X PBS, add 10 % donkey serum, and 0.3% Triton X-100 detergent.</td>
		</tr>
		<tr>
			<td>Buffer A</td>
			<td>Homemade</td>
			<td></td>
			<td>To make 1L: Mix 8.8g NaCl, 1.2g Tris base, 500ul Tween-20, with 750mL ddH20. pH to 7.5 with HCl, and fill to 1L. Filter solution and store at 4C. Bring to RT before experimental use</td>
		</tr>
		<tr>
			<td>Buffer B</td>
			<td>Homemade</td>
			<td></td>
			<td>To make 1L: Mix 5.84g NaCl, 4.24g Tris base, 26g Tris-HCl with 500mL ddH20. pH to 7.5, and fill to 1L. Filter solution and store a 4C. Bring to RT before experimental use</td>
		</tr>
		<tr>
			<td>Cy5-conjugated AffiniPure donkey anti-goat</td>
			<td>Jackson Labs</td>
			<td>705-175-147</td>
			<td></td>
		</tr>
		<tr>
			<td>Detection Reagents Red</td>
			<td>Sigma- Aldrich</td>
			<td>DU092008-100RXN</td>
			<td>Kit containing: ligation solution stock (5X), ligase, amplification solution stock (5X) and polymerase.</td>
		</tr>
		<tr>
			<td>DuoLink PLA probe anti-mouse MINUS</td>
			<td>Sigma- Aldrich</td>
			<td>DU092004-100RXN</td>
			<td></td>
		</tr>
		<tr>
			<td>DuoLink PLA probe anti-rabbit PLUS</td>
			<td>Sigma- Aldrich</td>
			<td>DU092002-100RXN</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Goat polyclonal anti-PDX1 (clone A17)</td>
			<td>Santa Cruz</td>
			<td>SC-14664</td>
			<td>RRID: AB_2162373</td>
		</tr>
		<tr>
			<td>HEPES (1M)</td>
			<td>Life Technologies</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr>
			<td>MIN6 pancreatic cell line</td>
			<td>Gift from D. Stoffers</td>
			<td></td>
			<td>Mouse insulinoma cell line, utilized for cell-based assays.</td>
		</tr>
		<tr>
			<td>Mouse monoclonal anti-USP8 antibody (clone US872)</td>
			<td>Sigma- Aldrich</td>
			<td>SAB200527</td>
			<td></td>
		</tr>
		<tr>
			<td>Pap-pen</td>
			<td>Research Products International</td>
			<td>195505</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td></td>
			<td></td>
			<td>Use to seal antibody and probe solutions on your cells to prevent evaporation when using small solution volumes.</td>
		</tr>
		<tr>
			<td>PBT (phosphate buffered saline with triton)</td>
			<td>Homemade</td>
			<td></td>
			<td>To make 50mL: 43.5mLddH2O, 5mL 10X PBS, 0.5mL 10X BSA(100mg/mL solution), 1mL 10% triton X-100 solution in ddH20)</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (100X)</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline, 10X</td>
			<td>Fisher Scientific</td>
			<td>BP399-20</td>
			<td></td>
		</tr>
		<tr>
			<td>PIM(ABS) Human AB serum</td>
			<td>Prodo Labs</td>
			<td>PIM-ABS001GMP</td>
			<td></td>
		</tr>
		<tr>
			<td>PIM(G) (glutamine)</td>
			<td>Prodo Labs</td>
			<td>PIM-G001GMP</td>
			<td></td>
		</tr>
		<tr>
			<td>PIM(S) media</td>
			<td>Prodo Labs</td>
			<td>PIM-S001GMP</td>
			<td></td>
		</tr>
		<tr>
			<td>PR619</td>
			<td>Apex Bio</td>
			<td>A812</td>
			<td></td>
		</tr>
		<tr>
			<td>Prolong Gold antifade reagent with DAPI</td>
			<td>Life Technologies (Molecular Probes)</td>
			<td>P36935</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit polyclonal anti-FLRF/RNF41 (Nrdp1)</td>
			<td>Bethyl Laboratories</td>
			<td>A300-049A</td>
			<td>RRID: AB_2181251</td>
		</tr>
		<tr>
			<td>SH-SY5Y cells</td>
			<td>Gift from L. Satin</td>
			<td></td>
			<td>Human neuroblastoma cell line, utilized for cell-based assays.</td>
		</tr>
		<tr>
			<td>Sodium Pyruvate (100X)</td>
			<td>Life Technologies</td>
			<td>11360-070</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher Scientific</td>
			<td>BP151-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Fisher Scientific</td>
			<td>BP337-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Water for RNA work (DEPC water)</td>
			<td>Fisher Scientific</td>
			<td>BP361-1L</td>
			<td></td>
		</tr>
	</tbody>
</table>

visualization of endogenous mitophagy complexes in situ in human pancreatic beta cells utilizing proximity ligation assay

Mitophagy 是一种重要的线粒体质量控制途径,对于胰腺胰岛β细胞生物能量促进葡萄糖刺激的胰岛素释放至关重要。评估米托巴经具有挑战性,通常需要基因报告员或多种补充技术,这些辅助技术不易用于组织样本,如原发性人类胰腺胰岛。在这里,我们演示了一种强大的方法来可视化和量化主要人类胰腺胰岛中关键内源性米托巴细胞复合物的形成。利用灵敏的接近结扎检测技术来检测米托巴风调节器NRDP1和USP8的相互作用,我们能够具体量化原位基本米托巴结物的形成。通过结合这种方法来对抗转录因子PDX1的染色,我们可以量化米托巴结物复合物,以及可能损害米托巴的因子,特别是在β细胞内。我们描述的方法克服了其他蛋白质-蛋白质相互作用研究(如免疫沉淀 (IP) 或质谱仪)所需的大量细胞提取物的需求,并且通常不适合珍贵的人类胰岛样本,通常不为这些方法提供足够数量的产品。此外,该方法消除了对流分选技术的需求,从异质小岛种群中纯化β细胞,用于下游蛋白质应用。因此,我们描述了一种有价值的方案,用于可视化米托巴细胞,高度兼容,用于异质和受限细胞群。

我们在MIN6胰腺β细胞系和SH-SY5Y神经母细胞瘤细胞系SH-SY5Y进行了初步实验,以优化和确认抗体的特异性和可视化的蛋白质相互作用。MIN6细胞以30,000个细胞/mL镀在盖玻片上,并留下粘附48小时,SH-SY5Y细胞以15,000个细胞/mL镀在盖玻片上,然后留在附着24小时。然后,从步骤 1.2.3 开始,遵循了上述 PLA 协议。为了确保PLA信号的特异性,我们首先在(i)没有原抗体(图2A,<st...

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Visualization of Endogenous Mitophagy Complexes In Situ in Human Pancreatic Beta Cells Utilizing Proximity Ligation Assay

利用邻近结扎测定法在人类胰腺β细胞中原位内源性米托巴结物的可视化

该协议概述了一种定量分析从原人类小岛样本的β细胞中专门形成米托巴细胞蛋白复合体的方法。因此,该技术允许从有限的生物材料分析米托巴,这在珍贵的人类胰腺β细胞样本中至关重要。

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated ...

该协议使我们能够监测人体组织中（如β细胞）的内源性食细胞复合物，促进关键转化相关组织中的线噬细胞研究。这种技术的一个优点是它能够可视化体内重要的食细胞复合物，在组织中具有罕见的可用性或小样本数。按照标准协议进行隔离后，在10毫升完整胰岛培养基中培养4至6倍10至第三个人类小岛，在37摄氏度下至少一天。第二天，使用三倍放大的解剖光显微镜来计算文化中的单个人类小岛。每次治疗或感兴趣的情况选择40个小岛，放入1.5毫升的小岛介质管中。通过离心沉淀小岛，将颗粒重新悬浮在PBS的一毫升中，并辅以50微摩尔PR619。反转管子，通过离心收集小岛，在新鲜的PBS加脱比酶抑制剂中进行第二次洗涤。第二次离心后，用125微升0.25%的三普辛加抑制剂将小岛分离成单细胞，在37摄氏度下进行三分钟，定期温和移液。在孵化结束时，用一毫升37摄氏度加热胰岛介质辅以PR619来阻止反应。通过离心收集小岛，在新鲜 PBS 中洗两次，每次洗涤使用抑制剂。第二次洗涤后，在150微升新鲜PBS加抑制剂中重新暂停分离的小岛。对于单个小岛粘附和固定，细胞将细胞溶液固定到带磨砂的显微镜幻灯片上，并用疏水笔勾勒出细胞区域。然后在室温下用4%甲醛和PBS将包围的细胞固定15分钟。对于小岛样品的免疫组织化学分析，在室温下用两次五分钟在PBS中清洗细胞，然后用10%驴血清和PBS加0.3%洗涤剂洗涤细胞，在室温下洗涤一小时。在阻断孵育结束时，用原发性小鼠抗体对泛素特异性肽酶8、原发性兔子抗体对neuregulin受体降解蛋白-1进行结合，并结合小鼠或兔子中未培养的β细胞识别标记，以便不干扰在4摄氏度过夜的接近结扎检测信号。第二天早上，用两个五分钟的PBS洗涤样品在摇杆上。将抗山羊Cy5二级抗体加入新准备的接近结扎测定溶液。第二次洗涤后，用20微升探针溶液给每个小岛样品贴上标签，用塑料薄膜回收幻灯片，在37摄氏度下孵育一小时。在孵育结束时，在摇杆上用两个五分钟的缓冲器A洗涤细胞，然后用20微升新鲜准备的结扎溶液进行孵育，每幻灯片每微升连接0.25个单位，在37摄氏度下孵育30分钟。在结扎结束时，用缓冲器 A 清洗细胞两次，每次洗涤两分钟。用 20 微升的扩增溶液覆盖每个样品，并辅以聚合酶，在 37 摄氏度下进行 100 到 120 分钟的孵育。要准备细胞进行成像，在摇杆上每次清洗时在新鲜缓冲液 A 中清洗两次样品 10 分钟，然后用 0.1 倍缓冲液 B 清洗一次，在摇杆上清洗两分钟。最后一次洗涤后，用一滴含有DAPI的安装介质安装幻灯片，并使用手术刀在每个样品上小心地放置盖玻片，以压出任何气泡。然后密封盖玻片，边缘周围有清晰的指甲油。要对样品进行成像，请将幻灯片放在能够捕获多个焦平面的显微镜的舞台上，然后选择 100 倍放大倍率。将 Z 堆栈高度设置为大约 0.45 微米，并设置适当的污渍、反染色和活细胞信号激发和发射参数。获取至少九个不同的焦平面图像。为确保荧光图像背景信号和杂散光最小化，使用选择的标准图像处理软件使用二维反卷积近邻算法处理图像。要量化接近结扎测定相互作用，请打开 ImageJ 并打开接近结扎测定图像。从第一张大幅对焦图像开始，单击"图像"并选择"调整"和"阈值"。调整阈值以删除所有非特定接近结扎分析信号，并记下阈值调整，以尝试在整个分析过程中保持信号一致。单击"进程"并选择二进制和"二进制"。确认"大小"设置为"大小"设置为无穷大，"循环"设置为零到 1，"显示"设置为"轮廓"并选中"显示结果"和"清除结果"框。请注意在电子表格中分析的表示样本和 Z 堆栈位置的粒子数。然后单击"分析和分析粒子"以测量粒子。分析样本的所有对焦 Z 堆栈后，发送电子表格中样本的粒子总数以量化交互总数。近位测定中使用的抗体是配体特异性的，不表现出任何重叠。正如所观察到的，分散协议在显微镜视野中获取单个细胞进行下游分析是高效的，在原人类小岛接近结扎测定染色后保留β细胞染色。利用所展示的技术，可以确定，在48小时接触棕榈酸盐后，神经素受体降解蛋白-1和泛素特异性肽酶8的相互作用减少，突出了这种测定的可行性，用于评估糖尿病刺激后的关键内源性三叶草因子。人类小岛高效而温和的分散对于确保下游正确细胞群的定量至关重要。PLA 允许评估人类胰岛样本中的重要线粒体复合物，从而能够分析线粒体和具有生物学重要性人类患者样本，从而推动糖尿病研究领域向前发展。

Research

JoVE Journal

Medicine

Staining Protocols for Human Pancreatic Islets

人类胰岛染色协议

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and ...

科研

医学

Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells

Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells

肺转移瘤在小鼠体内的组合在单细胞分辨率的可视化改进<em&gt;在原位</em灌注肺组织和X-gal染色<em&gt; lacZ的</em&gt;标记的肿瘤细胞

Metastasis is the main cause of death in the majority of cancer types and consequently a main focus in cancer research. However, the detection of ...

Coculture Analysis of Extracellular Protein Interactions Affecting Insulin Secretion by Pancreatic Beta Cells

共培养影响胰腺β细胞的胰岛素分泌细胞外蛋白质相互作用分析

Interactions between cell-surface proteins help coordinate the function of neighboring cells. Pancreatic beta cells are clustered together within ...

A Protocol for Genetic Induction and Visualization of Benign and Invasive Tumors in Cephalic Complexes of Drosophila melanogaster

A Protocol for Genetic Induction and Visualization of Benign and Invasive Tumors in Cephalic Complexes of Drosophila melanogaster

一个协议，用于基因诱导和良性和浸润性肿瘤可视化的头侧配合<em&gt;果蝇</em

Drosophila has illuminated our  understanding of the genetic basis of normal development and disease for the  past several decades and today it continues ...

Simulating Pancreatic Neuroplasticity: In Vitro Dual-neuron Plasticity Assay

Simulating Pancreatic Neuroplasticity: In Vitro Dual-neuron Plasticity Assay

模拟胰腺神经可塑性：<em&gt;体外</em&gt;双神经元可塑性分析

Neuroplasticity is an inherent feature of the enteric nervous system and gastrointestinal (GI) innervation under pathological conditions. However, the ...

Multi-electrode Array Recordings of Human Epileptic Postoperative Cortical Tissue

人类癫痫术后皮层组织的多电极阵列录音

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

研究胰腺癌干细胞特性开发新的治疗策略

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

从人类肿瘤异种移植的小鼠细胞的消耗显着改善靶细胞下游分析

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

体外和体内检测人类细胞中的 Mitophagy,线虫和小鼠

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...

Application of End-to-end Anastomosis in Robotic Central Pancreatectomy

端到端吻合技术在机器人中心胰腺中的应用

Central pancreatectomy is carried out for the treatment of benign or low-malignant potential tumors located in the pancreatic neck or proximal part of ...