Comparison of plate reader-based methods with fluorescence microscopy for measurements of intracellular calcium levels for the assessment of in vitro neurotoxicity

摘要

The intracellular calcium concentration ([Ca2+](i)) is an important readout for in vitro neurotoxicity since calcium is critically involved in many essential neurobiological processes, including neurotransmission, neurodegeneration and neurodevelopment. [Ca2+](i) is often measured with considerable throughput at the level of cell populations with plate reader-based assays or with lower throughput at the level of individual cells with fluorescence microscopy. However, these methodologies yield different quantitative and qualitative results. In recent years, we demonstrated that the resolution and sensitivity of fluorescence microscopy is superior compared to plate reader-based assays. However, it is currently unclear if the use of plate reader-based assays results in more 'false negatives' or 'false positives' in neurotoxicity screening studies. In the present study, we therefore compared a plate reader-based assay with fluorescence microscopy using a small test set of environmental pollutants consisting of dieldrin, lindane, polychlorinated biphenyl 53 (PCB53) and tetrabromobisphenol-A (TBBPA). Using single-cell fluorescence microscopy, we demonstrate that all test chemicals reduce the depolarization-evoked increase in [Ca2+](i), whereas lindane, PCB53 and TBBPA also increase basal [Ca2+](i), though via different mechanisms. Importantly, none of these effects were confirmed with the plate reader-based assay. We therefore conclude that standard plate reader-based methods are not sufficiently sensitive and reliable to measure the highly dynamic and transient changes in [Ca2+](i) that occur during chemical exposure. (C) 2014 Elsevier Inc. All rights reserved.