"Calcium Buffering by Fluorescent Indicators – Implications and Easy Solutions"

Intracellular free calcium concentrations ([Ca2+]i) are commonly measured with fluorescent indicators. As Ca2+ chelators, they compete with endogenous buffers for Ca2+ ions, which may reduce [Ca2+]i. Indicator concentrations that are unlikely to markedly affect Ca2+ signals were defined long time ago, but they are usually too low to allow accurate fluorescence measurements. In consequence, most routine [Ca2+] i measurements utilize much higher indicator concentrations and may underestimate [Ca2+]i. Here, we propose a method to derive correct, indicator-independent) [Ca2+]i by extrapolating the reported [Ca2+]i to the indicator-free environment. Although similar to its predecessors, the proposed steady-state model avoids complexities of earlier approaches and requires only data collected in AM ester loaded cells.