A robust fluorescence-based assay for human erythrocyte Ca++ efflux suitable for high-throughput inhibitor screens.
A robust fluorescence-based assay for human erythrocyte Ca++ efflux suitable for high-throughput inhibitor screens.
复制标题
一种基于荧光的人红细胞 Ca 流出检测方法，适用于高通量抑制剂筛选。
DOI：
10.1007/s00249-022-01623-y
复制
发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Desai,SanjayA
中科院分区：
文献类型：
--
作者：
Sims,JeremiahN;Yun,EJun;Chu,Jonathan;Siddiqui,MansoorA;Desai,SanjayA
细胞内钙通过PMCA Ca++挤出泵的作用维持在非常低的浓度。虽然我们的知识，这些Ca++挤出泵来自人类红细胞的研究，这些细胞的Ca++运输的动力学研究仅限于放射性同位素通量测量。在这里，我们开发了一个强大的，基于微孔板的测定红细胞Ca++流出使用细胞外荧光Ca++指标。我们优化了Ca++负载与A23187离子载体，建立了条件，去除离子载体，并调整荧光染料的灵敏度，通过添加细胞外EGTA，以允许连续跟踪Ca++流出。外排动力学加速葡萄糖和抑制剂量依赖性的方式由非特异性抑制剂钒酸盐，揭示钙++泵活性可以跟踪在384孔微孔板格式。这些研究使无放射性同位素动力学测量的Ca++泵，并应促进筛选这种基本的运输活动的特定抑制剂。
Intracellular calcium is maintained at very low concentrations through the action of PMCA Ca++extrusion pumps. Although much of our knowledge about these Ca++extrusion pumps derives from studies with human erythrocytes, kinetic studies of Ca++transport for these cells are limited to radioisotope flux measurements. Here, we developed a robust, microplate-based assay for erythrocyte Ca++efflux using extracellular fluorescent Ca++indicators. We optimized Ca++loading with the A23187 ionophore, established conditions for removal of the ionophore, and adjusted fluorescent dye sensitivity by addition of extracellular EGTA to allow continuous tracking of Ca++efflux. Efflux kinetics were accelerated by glucose and inhibited in a dose-dependent manner by the nonspecific inhibitor vanadate, revealing that Ca++pump activity can be tracked in a 384-well microplate format. These studies enable radioisotope-free kinetic measurements of the Ca++pump and should facilitate screens for specific inhibitors of this essential transport activity.