Calcium probes

Stability of aequorin. Information on the stability of aequorin is important when using this photoprotein as a calcium probe. As already noted, aequorin is always emitting a low level of luminescence, thus... 

This probe utilizes dual excitation wavelengths and is observed at a single emission wavelength. The intracellular calcium probes Fluo3 and Rhod2, which have recently been introduced, have fluorescein-like and rhodamine-like spectral properties, respectively (Minta, A. Harootunian, A.T. Kao, J.P.Y Tsien, R.J. J. Cell Biol 1987, 105,... 

Figure 1. Structures of the fluorescent calcium probes indo-1 and chlorotetracycline.

The difficulties of intensity-based flow cytometry are illustrated by the present difficulties of cell-by-cell measurements of intracellular calcium. This can be accomplished using the calcium probe Indo-l,(34 38) but requires an ultraviolet (UV) laser source which is not routinely available in flow cytometry (Indo-1 is an emission wavelength ratiometric probe). Flow cytometers routinely have argon ion laser sources with outputs of 488 or 514 nm. Measurement of intracellular ions other than Ca2+ is nearly impossible. (The SNAFL and SNARF probes should allow pH measurement from the wavelength-ratiometric data.(15))... 

E. U. Akkaya and J. R. Lakowicz, Styryl-based wavelength ratiometric probes A new class of fluorescent calcium probes with long wavelength emission and a large stokes shift, Anal. Biochem. 213, 285-289 (1993). 

In biomedical applications, the ranges of ion concentration are higher by several orders of magnitude. For instance, the abovementioned calcium probes for living cells cannot be used because the dissociation constant is so low that they would be saturated. Special attention is thus to be paid to the ionophore moiety to achieve proper selectivity and efficiency of binding. For instance, at present there is a need for a selective fluorescent probe for the determination of calcium in blood which could work in the millimolar range in aqueous solutions so that optodes with immobilized probes on the tip could be made for continuous monitoring calcium in blood vessels. 

The increase in phosphoinositide hydrolysis appears to occur during the G0 and early G[ phases of the cell cycle. Cells will not tolerate a sustained increase in intracellular Ca2+ and actively extrude the ion. Recent evidence in B lymphocytes, using calcium probes and image intensifies, shows that when surface immunoglobulin is crosslinked the rise of intracellular Ca2+ occurs in repeated short bursts [37]. 

Many of these dyes and their applications have been reviewed in detail elsewhere (50). Of note, probes are available for both UV and 488 nm excitation and many of these calcium probes (Fura-l, Indo-1, Calcium Green-2) have the advantage of utilizing a ratiometric fluorescence read-out. These dyes have been successfully used in multiparametric analyses, using both flow cytometry and confocal microscopy, which has led to an enhanced understanding of the role of [Ca2+]i in apoptosis. Burchiel et al. (51) have recently published a review of multiparametric flow cytometric Ca2+ analysis. 

Table II. Lifetimes and phase-modulation characteristics of the calcium probes...

Figure 12. Ca -dependent phase angles for calcium probes. Experimental condition are in Table II.

Intensity-Based Sensing. There are a number of probes which display changes in inlensicy but do not display spectral shifts. Such probes include the calcium probes Calcium Green . Fluo-3, and Rhod-2. In these cases, the analyte concentration can be obtained finsn... 

Calcium probes (Figure 19.49) are perhaps the most widely us l inbacellular indicators. All these indicators... 

Like the Na and K probes, Pura-2 and Indo-1 absorb in the UV. This is disadvantageous because, in addition to problems of cellular autofluorescence, it is difficult to obtun microscope optics with high UV transmission. Hence, it is desirable to develop calcium (Kobes with longer excitation and emission wavelengths. Coumarin- and styiyl-based calcium probes have been developed but have not yet been widely used. Excitation spectra of one such probe are shown in Figure 19.51. These probes allow excitation up to 520 nm, but wavelength-ratiometric measurements require a second excitation wavelength below 430 nm. 

Green series all increase on Ca binding, alloanng the calcium concentration to be determined ftom the Sfe-times. One of the first calcium probes, Quin-2, also displays a 10-fold increase in lifetime when boimd to... 

Althougli the use of the calcium probes seems stnugfit-finward. calibration is difficult when such probes are located within cells. " Reference 141 should be consulted fin a detailed description of the calibration procedures. When Used as iniracdlular indicalDrs, tbecalcium probes are typically calibrated in the presence of otlra intracellular ions at thdr expected concentrations, Also, it is difficult to maintain nanomolar Ca concentrations. 

For completeness, we note that Ca probes have also been developed using azacrown ethers as the chelator, rather than BAPTA." However, these probes have been studied mostly in organic solvents and used to study the effects of Cif on dectron transfer. Such probes have not found use in cell physiology. Magnesium-sensitive probes are also available (Table 19.4), " and some have been characterized as lifetime probes. These probes typically have the AFTRA chdator, father than BAFTA, as can be seen for the calcium probe Indo-1 and the analogous magnesium probe Mag-Indo-1 (Hguie 19.52). 

Brezden, C. B. Hedley, D. W. Rauth, A. M. Constitutive expression of P-glycoprotein as a determinant of loading with fluorescent calcium probes. Cytometry 1994, 17, 343-348. 

Haase, H. Hebei, S. Engelhard , G. Rink, L. Zinc ions cause the thimerosal-induced signal of fluorescent calcium probes in lymphocytes. Cell Calcium 2009, 45, 185-191. 

Mikkelson, R.B., 1976, Lanthanides as Calcium Probes in Membranes, in Biological Membranes, Vol. 3, eds D. Chapman and D.F. Wallach (Academic Press, New York) pp. 153-190. 

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