Ratiometric fluorescent calcium indicator dyes have been widely used for the study of the role of Ca2+ in cell physiopathology. Although these ratiometric dyes offer several advantages over others, they suffer some drawbacks which cause serious errors in measurement of intracellular Ca2+ concentration, [Ca2+]i. The present study systematically analyzes theoretical reasons and technical sources of discrepancies occurring in the measurement of the characteristics of the agonists-induced cells [Ca2+]i. In order to avoid the errors and achieve the accurate determination of [Ca2+]i, this study proposes solutions and suggests some critical measures in both theoretical and technical aspects. Therefore, this analysis can be a valuable tool in clarifying proper usages of fluorescent dyes for [Ca2+]i measurements.
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