Calibration Protocols for Fluorescent Calcium Indicators: Figure 1.

INTRODUCTION Fluorescent calcium indicators are useful for measuring intracellular calcium ion concentrations. For a quantitative understanding of the physiological roles of Ca 2+ , fluorescence signals measured with calcium indicators have to be converted to intracellular free calcium concentration ([Ca 2+ ] i ). Most methods for converting a fluorescence signal to [Ca 2+ ] i require the determination of a set of three calibration parameters: ( K eff , R min , R max ), ( K d , Δ F / F max , [Ca 2+ ] rest ), or ( K d , R f , F max ) or ( K app , τ min , τ max ). Here we describe the classical procedure for calibration of ratiometric measurements for both in vivo and in vitro calibrations, which is also useful for determining K d and R f . The [Ca 2+ ] i dependence of the fluorescence ratio is measured using a set of at least three calibration solutions with known [Ca 2+ ] i levels.