Caveats and limitations of plate reader-based high-throughput kinetic measurements of intracellular calcium levels

Calcium plays a crucial role in virtually all cellular processes, including neurotransmission. The intracellular Ca2+ concentration ([Ca2+]i) is therefore an important readout in neurotoxicological and neuropharmacological studies. Consequently, there is an increasing demand for high-throughput measurements of [Ca2+]i, e.g. using multi-well microplate readers, in hazard characterization, human risk assessment and drug development. However, changes in [Ca2+]i are highly dynamic, thereby creating challenges for high-throughput measurements. Nonetheless, several protocols are now available for real-time kinetic measurement of [Ca2+]i in plate reader systems, though the results of such plate reader-based measurements have been questioned. In view of the increasing use of plate reader systems for measurements of [Ca2+]i a careful evaluation of current technologies is warranted. We therefore performed an extensive set of experiments, using two cell lines (PC12 and B35) and two fluorescent calcium-sensitive dyes (Fluo-4 and Fura-2), for comparison of a linear plate reader system with single cell fluorescence microscopy. Our data demonstrate that the use of plate reader systems for high-throughput real-time kinetic measurements of [Ca2+]i is associated with many pitfalls and limitations, including erroneous sustained increases in fluorescence, limited sensitivity and lack of single cell resolution. Additionally, our data demonstrate that probenecid, which is often used to prevent dye leakage, effectively inhibits the depolarization-evoked increase in [Ca2+]i. Overall, the data indicate that the use of current plate reader-based strategies for high-throughput real-time kinetic measurements of [Ca2+]i is associated with caveats and limitations that require further investigation.

Research highlights
► The use of plate readers for high-throughput screening of intracellular Ca2+ is associated with many pitfalls and limitations.
► Single cell fluorescent microscopy is recommended for measurements of intracellular Ca2+.
► Dual-wavelength dyes (Fura-2) are preferred over single-wavelength dyes (Fluo-4) for measurements of intracellular Ca2+.
► Probenecid prevents dye leakage but abolishes depolarization-evoked Ca2+ influx, severely hampering measurements of Ca2+.
► In general, care should be taken when interpreting data from high-throughput kinetic measurements.