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TOOLS FOR CARDIOVASCULAR RESEARCH...

CONTRACTILITY AND RATIOMETRIC FLUORESCENCE SYSTEMS...

Calibration of Calcium Levels...

The ratiometric method of determining cell calcium levels using fura-2 is simple and elegant. One measures the fluorescence emission excited by two separate wavelengths, subtracts the background signal and the ratio between the intensity levels is used to calculate the free calcium concentration. The mathematical relationship between the measured ratio (R) and the calcium ion concentration is expressed as follows 1 ...

Ca = Kd (R-Rmin) / (Rmax - R) Sf2 / Sb2...

The evaluation of this expression requires the values for the so-called calibration constants (Kd, Rmax, Rmin, Sf2, and Sb2) to be inserted. The values of Rmax and Rmin are the ratio values measured under conditions of saturating calcium levels and in the absence of calcium respectively. The values of Sb2 and Sf2 are proportional to the fluorescence excited by the denominator wavelength (normally 380nm) again under conditions of saturating calcium levels (the "b" referring to the bound state) and in the absence of calcium (the "f" referring to calcium free) respectively. A value of 225nM is normally used for the dissociation constant for the fura2-Calcium binding (Kd). The ratio equation then generates calcium values in nanomolar. Careful determination of the calibration constants under conditions that closely match the actual experimental conditions is essential for obtaining reproducible calcium measurements. Since Rmax and Rmin derive from, and Sf2 and Sb2 essentially are fluorescence intensity values, their values depend on the characteristics specific optical elements in the system. These include the excitation and emission filters, dichroic mirrors and the microscope objective lens. Changing any optical element will have an effect on the calibration constants....

in vivo calibration...

There are two main strategies for determining the system-specific calibration constants. The conceptually simpler is the in vivo method. The notion here is to use the actual cells under investigation to assess the calibration values. To accomplish this one uses the normal loading procedure for fura2. It is then necessary to control the intracellular calcium concentration so that it can be set to essentially zero (to determine Rmin) and to a level that saturates the indicator (to determine Rmax). Controlling intracellular calcium levels is normally attempted using calcium ionophores (i.e. 8Bromo-A23187...

Document Keywords

Calcium Calibration userfiles file database calcium calibration

About IONOPTIX CORPORATION

Founded in 1990, IonOptix produces ratiometric fluorescence and cell dimensioning data acquisition systems for life scientists.

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