Rapid Measurements of Intracellular Calcium Using a Fluorescence Plate Reader

Intracellular calcium is a universal second messenger that can serve as a broadbased measure of receptor activity. Recent developments in multi-well plate fluorescence readers facilitate measurement of intracellular free-calcium levels and reduce reliance on slower, more cumbersome or expensive data collection methods. In this report, we describe a rapid and sensitive method to assay intracellular calcium ions in human embryonic kidney (HEK293) and Chinese hamster ovary (CHO) cells from multi-well plates using a fluorometer equipped with on-line injectors. We examine the compatibility of visible-light excitable dyes Calcium Green™-1 and Oregon Green™ 488 BAPTA-1. Using this assay, we were able to detect and quantify activity from muscarinic and β-adrenergic receptors endogenous to HEK293 cells and detect calcium signals generated by activation of Gi-coupled recombinant μ-opioid and dopamine D2L receptors, and the Gscoupled melanocortin subtype 4 (MC4) receptor. Fluorescence signals, stable in HEK293 cells, required the use 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 concentrationresponse data for a variety of receptors (including a recombinant muscarinic M1 receptor expressed in CHO cells) from a single plate of dye-loaded cells.