Reagents for Reporting Changes in Calcium Concentrations

Choosing a Dye With the Optimal Affinity for Calcium

It’s important to match the dye affinity for Ca²⁺ to the anticipated magnitude of the increase in calcium levels being imaged. For example, a high-affinity dye used to detect a large increase in Ca²⁺ will likely become saturated, resulting in an incorrect measurement of the magnitude of change [1]. Conversely, a low-affinity dye will likely be unable to detect small changes in Ca²⁺.

Dye emission intensities are also important to consider when choosing a dye for a particular imaging application. The Fluo family of dyes has a very low emission at rest, resulting in a large increase in emission intensity upon Ca²⁺ binding but making it difficult to resolve the cell at rest [1,2]. Members of the Oregon Green® 488 BAPTA dye family have a higher resting fluorescence and can therefore be used to locate cells and structures; however, the maximal change in emission intensity is lower upon Ca²⁺ binding.

High-affinity dyes such as Fluo-4, Fluo­-3, and Oregon Green® 488 BAPTA-1 and BAPTA-2 are best suited for detecting a response to low-intensity stimulation, whereby a small number of plasma membrane or intracellular Ca²⁺ channels are recruited. A response to medium-intensity stimulation of cells—whereby many channels are recruited for prolonged periods of time—is best detected using medium-affinity dyes such as Fluo-5F. For high-intensity stimulation, low-affinity dyes such as Oregon Green® 488 BAPTA-5N and Fluo-4FF are most useful (Table 1).

Choose red Ca²⁺ dyes for imaging cells or tissues with high autofluorescence, for simultaneous imaging of a green fluorophore (Figure 1), or for studies of calcium signaling (Figure 2). As with green-emitting dyes, there are both high-affinity (Rhod-3 and X-Rhod-1) and low-affinity (X-Rhod-5F) red-emitting dyes.

Figure 2.Imaging intracellular Ca²⁺ signaling. tSA-201 cells expressing CellLight® Tubulin-GFP and CaV2.1 (P Type) calcium ion channels were loaded with Rhod-3 according to instructions. The cells were rinsed once and placed in a laminar flow system and imaged at 100 msec intervals with a standard TRITC filter. When the solution was switched from control (time 0; A) to buffer containing high (50 mM) potassium substituted for sodium, the cells rapidly depolarized, allowing calcium influx through the opened CaV ion channels (B, C). The FITC filter image (D) shows GFP tubulin expressed in the same cells.

Loading Calcium Dyes Into Cells

Acetoxymethyl ester (AM) derivatives of fluorescent Ca²⁺ indicators are the most common dye form used for loading into cells. The AM derivative can cross the plasma membrane, followed by cleavage of the AM moiety by endogenous esterases to produce a charged molecule that is trapped within the cell. For single-cell imaging or recording of electrophysiological data, the cell-impermeant salt form of the dye can be loaded through intracellular injection or dialysis.

Ca²⁺ concentration changes can be imaged in locations other than the cytoplasm. For example, the low-affinity Mag-Fluo-4 dye can record high Ca²⁺ concentrations, such as those in the endoplasmic reticulum (ER) [3], without becoming saturated. In contrast, the Ca²⁺ affinity of this dye is too low to report changes in cytoplasmic Ca²⁺ concentration. When using Mag-Fluo-4 to report changes in ER Ca²⁺ concentration, the dye can be washed out of the cytoplasm via mild permeabilization or patch pipette dialysis to ensure that the detected signal is due to Ca²⁺ located in the ER.

The combination of Mag-Fluo-4 AM with a red cytoplasmic dye such as Rhod-3 AM can be used to simultaneously image cytoplasmic and ER Ca²⁺ concentration changes. Rhod-2 localizes to mitochondria, so it can be used to report mitochondrial Ca2+ transients [4].  Lysosomal Ca²⁺ can be measured by bathing cells in the dextran-conjugated version of certain Ca2+ dyes [5]. Ratiometric Ca²⁺ reporters, such as the organic dyes Indo-1 and Fura-2, and the genetically encoded Premo™ Cameleon Ca²⁺ sensor, are useful for imaging Ca²⁺ changes localized in small, subcellular regions, or in cases where movement of the cell or tissue may give rise to false changes in dye emission.

Complete Range of Reagents for Calcium Imaging

For more information on the complete range of reagents available for calcium imaging applications, see Introduction to Ca²⁺ Measurements with Fluorescent Indicators in The Molecular Probes® Handbook.

1. Maravall M, Mainen ZF, Sabatini BL et al. (2000) Biophys J 78:2655–2667.
2. Gee K, Brown KA, Chen W-N U et al. (2000) Cell Calcium 2:97–106.
3. Park MK, Tepikin AV, Petersen OH et al. (2000) EMBO J 19:5729–5739.
4. Quintanilla RA, Matthews-Roberson TA, Dolan PJ et al. (2009) J Biol Chem 284:18754–18766.
5. Lloyd-Evans E, Morgan AJ, He X et al. (2008) Nat Med 11:1247–1255.