Ratiometric and nonratiometric Ca²⁺ indicators for the assessment of intracellular free Ca²⁺ in a breast cancer cell line using a fluorescence microplate reader

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• 作者：Robinson ; Jodie A. ; Jenkins ; Nerida S. ; Holman ; Nicola A. ; Roberts-Thomson ; Sarah J. ; Monteith ; Gregory R.
• 关键词：Calcium ; Fura-2 ; MCF-7 ; Screening
• 刊名：Journal of Biochemical and Biophysical Methods
• 出版年：2004
• 期刊代码：84_0165022x
• 类别：bio
• 出版时间：March 31, 2004
• 卷：58
• 期：3
• 页码：227-237
• 文件大小：315 K

摘要

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.