Fluorescent Dyes: 
Fluorescent Dyes  CalibrationCalibration process implies calculating K_{d} value for our particular experimental conditions. This value depends on loading/measuring conditions (temperature, medium, concentration and volume used) and also on cell line. If fluorescence is represented vs. calcium concentration, a nonlinear curve is obtained, so K_{d} is not easily calculated.
Note that highaffinity indicators get saturated very quickly, so very small changes in intensity are observed above 1 µM for K_{d} = 100 nM ([Ca^{2+}] = 10·K_{d}). This example also illustrates the importance of working at concentrations near K_{d} value. If equation 1 is used, equation 2 can be obtained using logarithms.
Graphical representation of log [(F  F_{min})/(F_{max}  F)]* vs. log [Ca^{2+}] can be adjusted with a linear regression (y = a·x+b), where b = log K_{d}. Automatic calculation can be done using Molecular Probes' K_{d} calculator (this equation can also be applied to solution measured with a cuvette and a fluorometer). To obtain F values, different extracellular calcium concentrations are used and cells are treated with an ionophore that equilibrates extracellular and intracellular concentration and fluorescence or ratio is recorded. As calcium ion is very common in our environment (it is present in glassware, reagents, etc.), calcium buffers are recommended to avoid artefacts (it is more important at lower calcium concentrations) during calibration experiments. Obtaining exact F values is not as easy as it might seem at first glance, so some other details have to be considered to obtain an accurate K_{d} for each particular experimental condition. Many factors can affect its value, so special care has to be taken to avoid misleading results.
See also more tips on calcium calibration.
* For ratiometric indicators, log [(R  R_{min})/(R_{max} R)·(F^{λ1}_{max}/F^{λ1}_{min})] has to be used instead. 


