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 (F_max) and minimum (F_min), as well as the dissociation constant (Kd) of the Ca²⁺-Dye reaction (model 1). An alternative equation does not need the F_min but requires the rate constants k_on and k_off (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 F_min: [Ca²⁺] = Kd(F - F_rest/F_max - F) + [Ca²⁺]_IR(F_max - F_rest/F_max - F), where [Ca²⁺]_IR is the resting [Ca²⁺]. If the classical calibration approach is followed, the F_min can be estimated from: F_min = F_rest - ([Ca²⁺]_IR(F_max - F_rest)/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 F_min (1.24 ± 0.11 A.U., n = 4). However, when we used the mathematically estimated F_min (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 F_min 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 F_min avoids the uncertainties associated with its experimental measurement.