Sims, Jeremiah N.Yun, EJunChu, JonathanSiddiqui, Mansoor A.Desai, Sanjay A.2024-07-162024-07-162023Sims JN, Yun E, Chu J, Siddiqui MA, Desai SA. A robust fluorescence-based assay for human erythrocyte Ca++ efflux suitable for high-throughput inhibitor screens. Eur Biophys J. 2023;52(1-2):101-110. doi:10.1007/s00249-022-01623-yhttps://hdl.handle.net/1805/42230Intracellular 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.en-USPublisher PolicyCa++ ATPase pumpCalciumFluorescence Ca++ indicatorsHuman erythrocytesOrganic anion transportersTransportArticle