title: Ratiometric and nonratiometric Ca2+ indicators for the assessment of intracellular free Ca2+ in a breast cancer cell line using a fluorescence microplate reader.
abstract: Transporters of Ca2+ are potential drug targets and Ca2+ is a useful signal in the assessment of G-protein-coupled receptor activation. Assays involving the assessment of intracellular Ca2+ using microplate readers most often use Ca2+ indicators which do not exhibit a spectra shift on Ca2+ binding (e.g. fluo-3). Indicators that do exhibit a spectral shift upon Ca2+ binding (e.g. fura-2) offer potential advantages for the calibration of intracellular Ca2+ 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 Ca2+ in adherent breast cancer cells using ratiometric and nonratiometric Ca2+ indicators. Our results demonstrate that both fluo-3 and fura-2 detect ATP dose-dependent increases in intracellular Ca2+ 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 Ca2+ 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.
authors.affiliation: The School of Pharmacy, The University of Queensland, 4072, St. Lucia, Queensland, Australia. J.Robinson@pharmacy.uq.edu.au
authors.forename: Jodie A
authors.initials: JA
authors.lastname: Robinson
authors.forename: Nerida S
authors.initials: NS
authors.lastname: Jenkins
authors.forename: Nicola A
authors.initials: NA
authors.lastname: Holman
authors.forename: Sarah J
authors.initials: SJ
authors.lastname: Roberts-Thomson
authors.forename: Gregory R
authors.initials: GR
authors.lastname: Monteith
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000814:Aniline Compounds; D001943:Breast Neoplasms / Q000737:chemistry* / Q000378:metabolism; D002118:Calcium / Q000032:analysis* / Q000378:metabolism; D002460:Cell Line; D005260:Female; D005456:Fluorescent Dyes; D016257:Fura-2; D006801:Humans; D007424:Intracellular Fluid / Q000737:chemistry; D014966:Xanthenes
chemical_list: D000814:Aniline Compounds; D005456:Fluorescent Dyes; D014966:Xanthenes; C059715:Fluo-3; D000255:Adenosine Triphosphate; D002118:Calcium; D016257:Fura-2