Introduction to Biotium's Fluorescent Calcium Indicators And Related Products

Calcium is an important intracellular messenger ion responsible for the activation and deactivation of numerous
biological events in cells. To study the role of calcium in these events, it is essential for one to be able to quantitatively monitor its concentration. The most widely used method of Ca²⁺ detection is by the use of fluorescent Ca²⁺ indicators, a technique pioneered by professor Roger Tsien and colleagues (Tsien, R. in Methods in Cell Biology, Vol. 30, Taylor, D.L. and Wang, Y-L, Eds., Academic Press (1989) pp. 127-156).

Biotium offers a number of Ca²⁺ indicators, including Fura-2, Indo-1, Fluo-3 and Rhod-2. These products are available in both the membrane-impermeant salt forms and the membrane-permeant AM ester forms. The salt forms of the indicators are water-soluble and can be loaded into cells via microinjection. The AM esters of the indicators are membranepermeant and thus can be loaded into cells by simple incubation of the cell or tissue preparation in a buffer containing the AM ester. Biotium also supplies Pluronic F-127, a mild nonionic detergent that can facilitate the loading of the AM esters. Pluronic F-127 is offered FREE with each order of any AM esters. The AM esters themselves do not bind Ca²⁺. However, once they have entered the cells, they are readily hydrolyzed by intracellular esterases into the parent Ca²⁺ indicators, thus becoming responsive to Ca²⁺.

The primary differences among Fura-2, Indo-1 Fluo-3 and Rhod-2 are in their Ca²⁺ dissociation constants (K_d) or Ca²⁺ response range, excitation/emission wavelengths, spectral shift, and relative fluorescent quantum yields. Therefore, you should select a Ca²⁺ indicator that best suits your need in consideration of your biological system, instrument settings and any other fluorescent probes that you may use at the same time. The Kd values can give you an estimate of the detectable Ca²⁺ concentration range, usually 0.1K_d to 10K_d. However, one should be cautious in using these in vitro determined K_d values as the K_d values in cells could differ considerably due to differences in ionic strength, pH, viscosity and Ca²⁺ buffering by intracellular lipids and proteins (Petr, M.J. and Wurster, R.D. Cell Calcium, 21, 233(1997)).

Table 1.1 Physical properties of fluorescent calcium indicators

1. Molecular weights are for the free acid form of indicators; 2. Indicators for which a pair of wavelengths are isted can be used to detect calcium ratiometrically; 3. Calcium dissociation constant measured at 22oC in pH 7.2 buffer; 4.Bis-fura-2 has similar calcium response to Fura-2 but with a 75% larger extinction coefficient.