Cal Red™ R525/650 potassium salt

Calcium calibration can be carried out by measuring the fluorescence intensity of the salt form (25 to 50 µM in
fluorescence microplate readers) of the indicators in solutions with precisely known free Ca2+ concentrations. Calibration
solutions can be used based on 30 mM MOPS EGTA Ca2+ buffer. In general, water contains trace amount of calcium ion. It
is highly recommended to use 30 mM MOPS + 100 mM KCl, pH 7.2 as buffer system. One can simply make a 0 and 39 µM
calcium stock solutions as listed below, and these 2 solutions are used to make a serial solution of different Ca2+
concentrations.

A. 0 µM calcium: 30 mM MOPS + 100 mM KCl, pH 7.2 buffer + 10 mM EGTA

B. 39 µM calcium: 30 mM MOPS + 100 mM KCl, pH 7.2 buffer + 10 mM EGTA + 10 mM CaCl2

To determine either the free calcium concentration of a solution or the Kd of a single-wavelength calcium indicator,
the following equation is used: 

[Ca]free = Kd[F ─ Fmin]/[Fmax ─ F]

Where F is the fluorescence intensity of the indicator at a specific experimental calcium level, Fmin is the
fluorescence intensity in the absence of calcium and Fmax is the fluorescence intensity of the calciumsaturated probe. 

The dissociation constant (Kd) is a measure of the affinity of the probe for calcium. The calcium-binding and
spectroscopic properties of fluorescent indicators vary quite significantly in cellular environments compared to calibration
solutions. In situ response calibrations of intracellular indicators typically yield Kd values significantly higher than in vitro
determinations. In situ calibrations are performed by exposing loaded cells to controlled Ca2+ buffers in the presence of
ionophores such as A-23187, 4-bromo A-23187 and ionomycin. Alternatively, cell permeabilization agents such as digitonin
or Triton® X-100 can be used to expose the indicator to the controlled Ca2+ levels of the extracellular medium.