Low Affinity Indicators (LowAff)
Low affinity (formerly designated as “FF”) versions enable studies where a large concentration of calcium is expected. They have two major advantages over their high affinity counterparts. They cause reduced buffering of intracellular calcium and reduced perturbation of calcium transients, and they allow for measurements of shorter-lived transients. In the past, scientists used APTRA-based magnesium analogs for this purpose, but encountered interference from the binding of magnesium ions. TEFLabs provides low affinity versions of BAPTA-based calcium ion indicators to eliminate contribution due to magnesium.a The dissociation constant (Kd) is sensitive to pH, temperature, viscosity, ionic strength, competing ions, and cellular interactions. This Kd was measured in simple aqueousbuffers as a guideline to the scientist, who should then calibrate the indicator the cells under study.
b Once the AM ester form permeates the cell membrane, intracellular non-specific esterases hydrolyze the AM ester to yield the indicator in its Ca2+ sensitive salt form.
Our low affinity calcium indicators, based on the effect of fluorines on lowering the affinity of the regular indicators, have an appropriate Kd in the range of 15 µM to 25 µM. The first low affinity indicators from TEFLabs were Fura-2FF (now Fura-2 LowAff), and Indo-1FF (now Indo-1 LowAff). Low Affinity versions are also available for Asante Calcium Red, Fluo-2, and Rhod-2. The general properties of these indicators can be found in Table 6.1 below.
TEFLabs offers the even lower affinity nitro versions of Fura-2 and Indo-1 on a custom synthesis basis.
For those who would still prefer to use the APTRA-based indicators, Furaptra (Fura-Mg) and Indo-1 Mg (Mag-Indo)are still availble from TEFLabs.
The low affinity Ca2+ indicator selectivity over Mg2+ is illustrated in Figure 6.3. Fura-2 (Figure 6.3a) is saturated at levels as low as 2 µM Ca2+. This saturation may be avoided by using the higher Kd Furaptra or Fura-2 LowAff (Fura-2 FF). However, Furaptra (Figure 6.3b) cannot distinguish Mg2+ from Ca2+, such that it reports a high mitochondrial (M) concentration of Ca2+. Fura-2 LowAff (Figure 6.3c) reports the correctly low mitochondrial concentration for Ca2+ because Mg2+ does not contribute to its fluorescence response.
FIG. 6.4 Fluorescence excitation traces (emission at 510 nm) of a Ca titration of Fura-2 LowAff