A robust fluorescence-based assay for human erythrocyte Ca++ efflux suitable for high-throughput inhibitor screens
dc.contributor.author | Sims, Jeremiah N. | |
dc.contributor.author | Yun, EJun | |
dc.contributor.author | Chu, Jonathan | |
dc.contributor.author | Siddiqui, Mansoor A. | |
dc.contributor.author | Desai, Sanjay A. | |
dc.contributor.department | Surgery, School of Medicine | |
dc.date.accessioned | 2024-07-16T07:37:37Z | |
dc.date.available | 2024-07-16T07:37:37Z | |
dc.date.issued | 2023 | |
dc.description.abstract | 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. | |
dc.eprint.version | Author's manuscript | |
dc.identifier.citation | Sims 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-y | |
dc.identifier.uri | https://hdl.handle.net/1805/42230 | |
dc.language.iso | en_US | |
dc.publisher | Springer | |
dc.relation.isversionof | 10.1007/s00249-022-01623-y | |
dc.relation.journal | European Biophysics Journal | |
dc.rights | Publisher Policy | |
dc.source | PMC | |
dc.subject | Ca++ ATPase pump | |
dc.subject | Calcium | |
dc.subject | Fluorescence Ca++ indicators | |
dc.subject | Human erythrocytes | |
dc.subject | Organic anion transporters | |
dc.subject | Transport | |
dc.title | A robust fluorescence-based assay for human erythrocyte Ca++ efflux suitable for high-throughput inhibitor screens | |
dc.type | Article |