A new set of equations for the simplified calibration of fluorescence Ca 2+ transients in skeletal muscle fibers

The classical approach for calibrating non-ratiometric fluorescent Ca 2+ dyes entails the measurement of the fluorescence maximum ( F max ) and minimum ( F min ), as well as the dissociation constant ( Kd ) of the Ca 2+ —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 Ca 2+ transients obtained with non-ratiometric dyes. This equation allows the calibration of signals without using the F min : [Ca 2+ ] = Kd ( F − F rest / F max − F ) + [Ca 2+ ] IR ( F max − F rest /F max − F ), where [Ca 2+ ] IR is the resting [Ca 2+ ]. If the classical calibration approach is followed, the F min can be estimated from: F min = F rest − ([Ca 2+ ] 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 [Ca 2+ ] 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 [Ca 2+ ] 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 [Ca 2+ ]. In conclusion, we propose simpler and time-saving equations that help to reliably calibrate cytosolic Ca 2+ transients obtained with non-ratiometric fluorescent dyes. The use of the estimated F min avoids the uncertainties associated with its experimental measurement.