A new set of equations for the simplified calibration of fluorescence Ca2+ transients in skeletal muscle fibers.

The classical approach for calibrating non-ratiometric fluorescent Ca2+ dyes entails the measurement of the fluorescence maximum (Fmax) and minimum (Fmin), as well as the dissociation constant (Kd) of the Ca2+-Dye reaction (model 1). An alternative equation does not need the Fmin but requires the rate constants kon and koff (model 2). However, both approaches are experimentally time consuming and the rate constants for several dyes are unknown. Here, we propose a set of equations (model 3) that simplify the calibration of fluorescent Ca2+ transients obtained with non-ratiometric dyes. This equation allows the calibration of signals without using the Fmin: [Ca2+] = Kd(F - Frest/Fmax - F) + [Ca2+]IR(Fmax - Frest/Fmax - F), where [Ca2+]IR is the resting [Ca2+]. If the classical calibration approach is followed, the Fmin can be estimated from: Fmin = Frest - ([Ca2+]IR(Fmax - Frest)/Kd). We tested the models' performance using signals obtained from enzymatically dissociated flexor digitorum brevis fibers of C57BL/6 mice loaded with Fluo-4, AM. Model 3 performed the same as model 2, and both gave peak [Ca2+] values 15 ± 0.3% (n = 3) lower than model 1, when we used our experimental Fmin (1.24 ± 0.11 A.U., n = 4). However, when we used the mathematically estimated Fmin (6.78 ± 0.2 A.U) for model 1, the peak [Ca2+] were similar for all three models. This suggests that the dye leakage makes a correct determination of the Fmin unlikely and induces errors in the estimation of [Ca2+]. In conclusion, we propose simpler and time-saving equations that help to reliably calibrate cytosolic Ca2+ transients obtained with non-ratiometric fluorescent dyes. The use of the estimated Fmin avoids the uncertainties associated with its experimental measurement.