Rapid measurements of intracellular calcium using a fluorescence plate reader

Intracellular calcium is a universal second messenger that can serve as a b road-based measure! of receptor activity. Recent developments in multi-well plate fluorescence readers facilitate measurement of intracellular free-ca lcium levels and reduce reliance an slower; more cumbersome or expensive da ta collection methods. In this report, we describe a rapid and sensitive me thod to assay intracellular calcium ions in human embryonic kidney (HEK293) and Chinese hamster ovary (CHO) cells from multi-well plates using a fluor ometer equipped with opt-line injectors. We examine the compatibility of vi sible-light excitable dyes Calcium Green(TM)-1 and Oregon Green(TM) 488 BAP TA-1. Using this assay, ne Ir ere able to detect and quantify activity from muscarinic and beta-adrenergic receptors endogenous to HEK293 cells and de tect calcium signals generated by activation of Gi-coupled recombinant mu-o pioid and dopamine D2L receptors, and the Gs-coupled melanocortin subtype 4 (MC4) receptor: Fluorescence signals, stable in HEK293 cells required the nse of Oregon Green 488 BAPTA-1 and an inhibitor of organic anion transport in CHO cells. Under appropriate conditions both cell types can be used to collect complete concentration-response data for a variety of receptors (in cluding a recombinant muscarinic MI receptor expressed in CHO cells) from a single plate of dye-loaded cells.