Abstract & Introduction

To measure the activity of Gq-coupled GPCRs in drug discovery and compound screening campaigns, intracellular calcium flux assays using fluorescent calcium
binding dyes play an important role. The FLIPR Tetra® system offers real-time kinetic measurement of calcium flux within cells and is traditionally performed in
384-well assay plates. However, as high-throughput screening campaigns have been increasing in size over the past years, the substantial number of cells and
reagents required, and the overall duration of the campaigns have become increasingly important restricting factors.

To circumvent these limitations, we have miniaturized a cellular calcium flux assay to 1536-well format. Using a FLIPR Tetra® equipped with a 1536-pipettor head,
we performed a mini-screen of 2,816 compounds from the Evotec-lead like compound library. We identified agonists and allosteric modulators of endogenously
expressed purinergic receptors in wild type CHO cells (WT CHO) showing dose-dependent activation, demonstrating suitability of the 1536-well format for calcium
flux assays in screening mode.

Our study demonstrates the advantages of miniaturization of a cell-based calcium flux assay into a 1536-well format using the FLIPR Tetra®, by gathering real
time data in high-throughput mode using much less cells, smaller reagent volumes and a significantly increased throughput time, thereby enabling time- and cost efficient screening of libraries with >250,000 compounds.