非人灵长类动物胰岛氧消耗分析
Analysis of Non-Human Primate Pancreatic Islet Oxygen Consumption.
作者信息
Elsakr Joseph M, Deeter Charles, Ricciardi Valerie, Gannon Maureen
机构信息
Department of Molecular Physiology and Biophysics, Vanderbilt University.
Agilent Technologies.
出版信息
J Vis Exp. 2019 Dec 18(154). doi: 10.3791/60696.
The measurement of oxygen consumption in spheroid clusters of cells, such as ex vivo pancreatic islets, has historically been challenging. We demonstrate the measurement of islet oxygen consumption using a 96-well microplate designed for the measurement of oxygen consumption in spheroids. In this assay, spheroid microplates are coated with a cell and tissue adhesive on the day prior to the assay. We utilize a small volume of adhesive solution to encourage islet adherence to only the bottom of the well. On the day of the assay, 15 islets are loaded directly into the base of each well using a technique that ensures optimal positioning of islets and accurate measurement of oxygen consumption. Various aspects of mitochondrial respiration are probed pharmacologically in non-human primate islets, including ATP-dependent respiration, maximal respiration, and proton leak. This method allows for consistent, reproducible results using only a small number of islets per well. It can theoretically be applied to any cultured spheroids of similar size.
对细胞球体簇（如离体胰岛）中的氧消耗进行测量，在历史上一直具有挑战性。我们展示了使用专为测量球体中的氧消耗而设计的96孔微孔板来测量胰岛氧消耗。在该测定中，在测定前一天，将球体微孔板用细胞和组织粘合剂包被。我们使用少量的粘合剂溶液来促使胰岛仅粘附于孔的底部。在测定当天，使用一种确保胰岛最佳定位和准确测量氧消耗的技术，将15个胰岛直接加载到每个孔的底部。在非人类灵长类动物胰岛中，通过药理学方法探究线粒体呼吸的各个方面，包括ATP依赖性呼吸、最大呼吸和质子泄漏。该方法仅使用每个孔少量的胰岛就能获得一致、可重复的结果。理论上，它可应用于任何类似大小的培养球体。
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