Lee Wing-Kee, Dittmar Thomas
Institute for Physiology, Pathophysiology, & Toxicology, Centre for Biomedical Research and Training (ZBAF), University of Witten/Herdecke;
Institute for Immunology & Experimental Oncology, Centre for Biomedical Research and Training (ZBAF), University of Witten/Herdecke.
J Vis Exp. 2014 Oct 28(92):e51857. doi: 10.3791/51857.
A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.
多种细胞过程，包括生理和病理生理过程，都需要钙或受钙调控，仅举几例，如胞吐作用、线粒体功能、细胞死亡、细胞代谢和细胞迁移。胞质钙通常维持在低纳摩尔浓度；相反，在内质网、线粒体基质和细胞外区室中，其浓度则处于高微摩尔至毫摩尔水平。受到刺激后，胞质钙的短暂增加用于向下游事件发出信号。检测胞质钙的变化通常使用配备钙结合染料的活细胞成像装置，这些染料在结合钙后会表现出荧光强度增加或发射波长偏移。然而，并非所有研究人员都能自由使用活细胞成像装置。针对免疫细胞，已优化了流式细胞术检测方法；针对非免疫贴壁细胞，已优化了荧光微孔板读数器检测方法。在此，我们描述一种优化的、简单的方法，使用单波长钙结合染料通过流式细胞术检测上皮细胞中的变化。在丙磺舒存在的情况下，用一种可透过细胞的荧光钙结合染料加载通常难以加载染料的贴壁肾近端小管上皮细胞，使其悬浮，然后在添加毒胡萝卜素、衣霉素和离子霉素之前和之后监测钙信号。
