- Highly sensitive dye for rapid measurement of calcium flux in cells
- Large fluorescence increase (typically >100-fold) provides good sensitivity
- Extensively used for cell-based HTS applications
- Visible light-excitable dye for less autofluorescence and cellular photodamage issues, and efficient excitation with most laser-based instrumentation, including confocal laser-scanning microscopes and flow cytometers
The Ca2+ indicator fluo-3 (Figure 1) was developed by Tsien and colleagues for use with visible-light excitation sources in flow cytometry and confocal laser-scanning microscopy. Since being introduced in 1989, fluo-3 imaging has revealed the spatial dynamics of many elementary processes in calcium signaling. Fluo-3 has also been extensively used for flow cytometry; for experiments involving photoactivation of “caged” chelators, second messengers, and neurotransmitters; and for cell-based pharmacological screening.
The most important properties of fluo-3 in these applications are an absorption spectrum compatible with excitation at 488 nm by argon-ion laser sources, and a very large fluorescence intensity increase in response to Ca2+ binding (Figure 2). Fluo-3, fluo-4 and their derivatives all exhibit large fluorescence intensity increases on binding Ca2+. Unlike the ultraviolet light-excited indicators fura-2 and indo-1, there is no accompanying spectral shift. The fluorescence intensity increase on Ca2+ binding is typically >100-fold. A comparison of physical and spectroscopic properties for fluo-3 and fluo-4 is shown in Table 1.
Table 1. Comparison of physical and spectroscopic properties for fluo-3 and fluo-4.
| Property | fluo-3 | fluo-4 |
|---|---|---|
| Kd (Ca2+)* | 325 nM | 345 nM |
| Absorption maximum (Ca2+-bound) † | 506 nm | 494 nm |
| max (Ca2+-bound) † | 100,000 cm-1M-1 | 88,000 cm-1M-1 |
| 488 nm (Ca2+-bound) † | 43,000 cm-1M-1 | 77,000 cm-1M-1 |
| Emission maximum (Ca2+-bound) † | 526 nm | 516 nm |
| QY (Ca2+-bound) †, ‡ | 0.15 | 0.14 |
| Fmax /Fmin § | >100 | >100 |
* Dissociation constant for Ca2+ determined at 22° C in 100 mM KCl, 10 mM MOPS, pH 7.2, 0 to 10 mM CaEGTA. Kd = 390 nM for fluo-3 reported in Molecular Probes’ The Handbook: A Guide to Fluorescent Probes and Labeling Technologies, Tenth Edition (2005).
† Value determined at 22° C in 100 mM KCl, 10 mM MOPS, pH 7.2 containing 39.8 μM free Ca2+.
‡ QY = fluorescence quantum yield. QY = 0.18 for fluo-3 reported in J Biol Chem 264, 8171 (1989).
§ Fluorescence intensity increase on binding Ca2+ in a solution assay.
For Research Use Only. Not for use in diagnostic procedures.