Fluorescent Dyes:

### Fluorescent Dyes - Calibration

Calibration process implies calculating Kd 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 non-linear curve is obtained, so Kd is not easily calculated.

 If fluorescence intensity changes with calcium concentration for different Kd values (an Fmax = 100 and Fmin = 0 are used in the graph) are analyzed, it is easy to see the non-linear relationship at high calcium values. Note that high-affinity indicators get saturated very quickly, so very small changes in intensity are observed above 1 µM for Kd = 100 nM ([Ca2+] = 10·Kd). This example also illustrates the importance of working at concentrations near Kd value.

If equation 1 is used, equation 2 can be obtained using logarithms.

 [Ca2+] = Kd · (F - Fmin)/(Fmax - F) Equation 1. log [(F - Fmin)/(Fmax - F)] = log [Ca2+] + log Kd Equation 2.

Graphical representation of log [(F - Fmin)/(Fmax - F)]* vs. log [Ca2+] can be adjusted with a linear regression (y = a·x+b), where b = log Kd. Automatic calculation can be done using Molecular Probes' Kd 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 Kd for each particular experimental condition. Many factors can affect its value, so special care has to be taken to avoid misleading results.