Ratiometric and nonratiometric Ca 2+ indicators for the assessment of intracellular free Ca 2+ in a breast cancer cell line using a fluorescence microplate reader

Abstract

Transporters of Ca 2+ are potential drug targets and Ca 2+ is a useful signal in the assessment of G-protein-coupled receptor activation. Assays involving the assessment of intracellular Ca 2+ using microplate readers most often use Ca 2+ indicators which do not exhibit a spectra shift on Ca 2+ binding (e.g. fluo-3). Indicators that do exhibit a spectral shift upon Ca 2+ binding (e.g. fura-2) offer potential advantages for the calibration of intracellular Ca 2+ levels. However, experimental limitations may limit the use of ratiometric dyes in microplate readers capable of screening. In this study, we compared the assessment of intracellular Ca 2+ in adherent breast cancer cells using ratiometric and nonratiometric Ca 2+ indicators. Our results demonstrate that both fluo-3 and fura-2 detect ATP dose-dependent increases in intracellular Ca 2+ in the MCF-7 breast cancer cell line and that some of the limitations in the use of fura-2 appear to be overcome by the use of glass bottom microplates. The calibrated intracellular Ca 2+ levels derived using fura-2 are consistent with those from microscopy and cuvette-based studies. Fura-2 may be useful in microplate studies, where cell lines with different properties are compared or where screening treatments lead to differences in the number of cells or dye loading.