Calibration Protocols for Fluorescent Calcium Indicators: Figure 1.

Abstract

INTRODUCTIONFluorescent calcium indicators are useful for measuring intracellular calcium ion concentrations. For a quantitative understanding of the physiological roles of Ca2+, fluorescence signals measured with calcium indicators have to be converted to intracellular free calcium concentration ([Ca2+]i). Most methods for converting a fluorescence signal to [Ca2+]i require the determination of a set of three calibration parameters: (Keff, Rmin, Rmax), (Kd, ΔF/Fmax, [Ca2+]rest), or (Kd, Rf, Fmax) or (Kapp, τmin, τmax). 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 Kd and Rf. The [Ca2+]i dependence of the fluorescence ratio is measured using a set of at least three calibration solutions with known [Ca2+]i levels.