Ho Kung-Hsien, Gu Guoqiang, Kaverina Irina
Department of Cell and Developmental Biology, Vanderbilt University, Nashville, TN, USA.
Program in Developmental Biology and Center for Stem Cell Biology, Vanderbilt University, Nashville, TN, USA.
Bio Protoc. 2023 Nov 5;13(21):e4868. doi: 10.21769/BioProtoc.4868.
Pancreatic islet β cells preferentially secrete insulin toward the plasma membrane, making contact with the capillary extracellular matrix (ECM). Isolated islets separated from the exocrine acinar cells are the best system for cell biology studies of primary β cells, whereas isolated islets lose their capillary network during ex vivo culture. Providing the appropriate extracellular signaling by attaching islets to vascular ECM-coated surfaces can restore the polarized insulin secretion toward the ECM. The guided secretion toward ECM-coated glass coverslips provides a good model for recording insulin secretion in real time to study its regulation. Additionally, β cells attached to the ECM-coated coverslips are suitable for confocal live imaging of subcellular components including adhesion molecules, cytoskeleton, and ion channels. This procedure is also compatible for total internal reflection fluorescence (TIRF) microscopy, which provides optimal signal-to-noise ratio and high spatial precision of structures close to the plasma membrane. In this article, we describe the optimized protocol for vascular ECM-coating of glass coverslips and the process of attachment of isolated mouse islets on the coverslip. This preparation is compatible with any high-resolution microscopy of live primary β cells. Key features • Optimized coating procedure to attach isolated islets, compatible for both confocal and TIRF microscopy. • The ECM-coated glass coverslip functions as the artificial capillary surface to guide secretion toward the coated surface for optimal imaging of secretion events. • Shows the process of islets attachment to the ECM-coated surface in a 6-day ex vivo culture.
胰腺胰岛β细胞优先向质膜分泌胰岛素，与毛细血管细胞外基质（ECM）接触。从外分泌腺泡细胞中分离出的孤立胰岛是原代β细胞细胞生物学研究的最佳系统，然而，孤立胰岛在体外培养过程中会失去其毛细血管网络。通过将胰岛附着在血管ECM包被的表面提供适当的细胞外信号，可以恢复向ECM的极化胰岛素分泌。向ECM包被的玻璃盖玻片的定向分泌为实时记录胰岛素分泌以研究其调节提供了一个良好的模型。此外，附着在ECM包被的盖玻片上的β细胞适用于对包括粘附分子、细胞骨架和离子通道在内的亚细胞成分进行共聚焦实时成像。该方法也适用于全内反射荧光（TIRF）显微镜，其提供了最佳的信噪比和靠近质膜结构的高空间精度。在本文中，我们描述了玻璃盖玻片血管ECM包被的优化方案以及分离的小鼠胰岛在盖玻片上的附着过程。这种制备方法适用于原代活β细胞的任何高分辨率显微镜检查。关键特性•优化的包被程序以附着分离的胰岛，适用于共聚焦显微镜和TIRF显微镜。•ECM包被的玻璃盖玻片作为人工毛细血管表面，引导向包被表面的分泌，以实现分泌事件的最佳成像。•展示了在6天体外培养中胰岛附着于ECM包被表面的过程。
