Quest Fluo-8® AM, Fluo-4 Direct™ and Calcium-4™ have been compared and evaluated for several GPCR and ion channel targets. Quest Fluo-8
® AM, Fluo-4™ and Calcium 4 share the same assay principle. All the three fluorogenic calcium indicators are in the form of non-fluorescent AM esters. Once inside cells, the lipophilic AM blocking groups are cleaved by non-specific cellular esterases, resulting in negatively charged fluorescent dyes that stay inside cells, and their fluorescence intensities are greatly enhanced upon binding to calcium. When cells stimulated with screening compounds, the receptor signals release of intracellular calcium, which greatly increases their fluorescence signals. Three GPCRs (Muscarinic M1 receptor, purinergic P2Y receptor, and chemokine CCR2b receptor) and one calcium channel capacitative calcium entry (CCE) were assayed. In comparison with Fluo-4™ Direct and Calcium 4, Quest Fluo-8
® yields the brightest signal and largest assay window. Quest Fluo-8
® AM has a less temperature-dependent cell loading property, giving more consistent results either at room temperature or 37°C compared to Fluo-4™ Direct or Calcium-4. This characteristic makes Quest Fluo-8
® AM much more robust for HTS applications.