Fluorescence indicator fura

Calcium ion fluxes mediated by a variety of channels and ionophores into liposomes and cells have been studied by loading the vesicles with indicator dyes like arsenazo III (129) or quin-2 (130). The significantly higher calcium affinity of the fluorescence indicator fura-2 (131) was a major advancement in the detection of Ca2+ concentrations in small cells and liposomes (132-134). 

Isolated chromaffin cells were maintained in suspension culture and loaded with the fluorescent calcium indicator Fura 2 as previously described (28). 2 x 10 cells/ml were added into a cuvette containing standard buffer without (dotted line) or with (full line) 2 mM calcium. At the arrow, 10" M pardaxin was added. A rise in was... 

M-5 (Quin-2) was the first practical fluorescent indicator for cytosolic calcium with a simple 6-methoxyquinoline as its fluorophore. Ca2+-binding increases the fluorescence intensity about six-fold (without spectral displacement, in contrast to Fura 2 see Section 10.3.3). The fluorescence lifetime of Quin-2 is highly sensitive to calcium concentration Quin-2 can thus be used as a probe in the technique of fluorescence lifetime imaging. 

Recently, Tsubosaka et al. (2010a) reported that halichlorine was also revealed to inhibit L-type Ca2+ channels, which leads to inhibit smooth muscle contraction. In their report, the direct effect of halichlorine on vascular contractility was investigated. Then, halichlorine was found to inhibit both high concentration of K+- and phenylephrine-induced contractions in rat aorta dose dependently. The effect of halichlorine on high K+-induced contraction was shown to be stronger than that on phenylephrine-induced contraction. Because known L-type Ca2+ channel blockers, verapamil and nifedipine, were observed to show the similar effect by them, it was suggested that halichlorine selectively inhibits L-type Ca2+ channels. Then, the effect of halichlorine on intracellular Ca2+ concentration in vascular smooth muscle tissue was examined using a fluorescent Ca2+ indicator, Fura-2. 

The method used for determination of PolyP, which is based on the Mn2+-induced quenching of the fluorescence of the calcium indicator Fura-2, has been described (Lorenz et al., 1997a). The effect of Mn2+ ions on the Fura-2 fluorescence is gradually removed in the presence of increasing PolyPs concentrations this allows the quantification of PolyPs isolated from tissues or cells. The described method has some advantages when compared with the conventional detection procedures based on the metachromatic effect. It can be applied to the determination of pyrophosphate, tripolyphosphate and other short-chain PolyPs not detectable by toluidine blue (Lorenz et al., 1997a). 

Earlier techniques for measuring cytosolic free Ca2+ (1-2) such as the luminescent photoprotein aequorin, the absorbance dye arsenazo III and Ca2+-sensitive microelectrodes, all required microinjection or impalements, and were therefore applied mainly to giant cells. Later, photoproteins have been loaded with various reversible permeabilization procedures (3), but the largest expansion in the range of cell types in which Ca2+ signals can be quantified has come from the development of new fluorescent indicators that can be loaded using hydrolyzable esters. Currently four fluorescent indicators are used frequently quin-2, fura-2, indo-1 and fluo-3. 

FIGURE 31.3 Effect of palytoxin on the cytosolic calcium concentration in primary cultured neurons loaded with the fluorescent calcium indicator Fura-2 AM. (Data extracted from Vale, C and Ares, I.R., in Phycotoxins Chemistry and Biochemistry, Botana, L. (Ed.), Blackwell Publishing, 2007, pp. 95-118.)... 

FIGURE 31.6 Effect of different MAPK inhibitors on the palytoxin-induced calcium influx in cultured neurons. Cerebellar granule cells were exposed to 10 nM palytoxin in the presence or absence of different MAPK inhibitors and the cytosolic calcium concentration was monitored with the fluorescent calcium indicator Fura-2 AM. 

D. discoideum have provided evidence for this transient increase. These include loading cells with the fluorescent Ca + indicator fura-2-dextran and imaging individual cells, measuring Ca uptake using the radioisotope and in vivo expression of... 

This approach to the imaging of intracellular Ca " flux was initiated by the work of Dr. Roger Tsien. At the University of California at Berkeley, he developed the Ca -selective chelator, BAPTA (2) and it s initial fluorescent derivatives. With the invention of quin-2 in 1980 (J) and Ae subsequent indicators, fura-2, indo-1 (4), fluo-3 and rhod-2 (5), researchers in the biological sciences have begun to define the complex role of Ca " in living cells (6). 

Fluorescent indicators for ions are based on corresponding chelators. The indicators usually mimic the structure of ethyleneglycol- / 5(P-aminoethylether)-A, A/,/V, /V -tetraacetic acid (EGTA), which also contains an adjacent fluorophore moiety affected by the binding. The most popular intracellular Ca " indicator, Fura-2, displays a shift in excitation maximum between 300-400 nm when monitoring emission at 510 nm, thus allowing ratiometric measurements. Calcium Green displays an increase in emission at 530 nm on addition of calcium but displays no spectral shift. Other fluorescent calcium indicators with different affinities and spectral characteristics are available that allow for the measurement of from nano- to submillimolar concentrations. 

Garcia-Martin, E. Martin-Romero, F. J. Gutierrez-Merino, C. Intrasynaptosomal free Mg " " concentration measured with the fluorescent indicator Mag-Fura-2 modulation by Na" " gradient and by extrasynaptosomal ATP. J. Neurochem. 1995, 65, 2757-2764. 

Tojyo, Y Tanimura, A. Matsumoto, Y. Monitoring of Ca release from intracellular stores in permeabilized rat parotid acinar cells using the fluorescent indicators Mag-Fura-2 and Calcium Green Cl8. Biochem. Biophys. Res. Common. 1997, 240, 189-195. 

Another strategy for monitoring Cd " in cells using BAPTA-based fluorescence indicators involves the use of two types of dyes that exhibit different fluorescent responses to Ca " and Cd ", respectively. As opposed to Fura-2, no change in the fluorescence of Quin-2, which is a tum-on type Ca " indicator based on a quinoline scaffold, is induced by adding Cd " [43]. By taking advantage of the different photophysical properties of Cd " complexes of Fura-2 and Quin-2, Cd " uptake by... 

Intracellular calcium elevation is monitored by fluorescent chelators developed by Tsien and coworkers. These indicators are loaded into cells the same way the pH indicators are. With Quin-2 (14), one of the first such probes developed, the quantum yield increases about fourfold when Ca binds to it. The second generation of Ca probes, Indo-1 and Fura-2 (15), are now being widely used in a variety of cell types. These probes are in most cases... 

FIG. 2. Simultaneous recording of membrane currents and Ca2+ fluorescence. (A) Upper and lower traces indicate the time courses of membrane current and [Ca2+] respectively. Cells were voltage-clamped at — 60 mV. Pipette contained Cs aspartate internal solution supplemented with 50 M fura-2. (B) Expanded time-courses of membrane current and [Ca2+] form the dotted box in A. TG, thapsigargin 2-APB, 2-aminoethoxydiphenyl borate. 

In contrast to Fura-2, the photoinduced charge transfer in Indo-1 may not be sufficient to cause nitrogen-Ca2+ bond breaking. This interpretation is consistent with the fact that the fluorescence maximum of free Indo-1 is located at a shorter wavelength than Fura-2 by 30 nm, thus indicating a less polar charge-transfer state. 

Most of the fluorescent calcium indicators and their cell-permeant acetoxymethyl (AM) esters are variations of the nonfluorescent calcium chelator BAPTA and have been proposed by Tsien/134-1365 Among them Fura-2 and Indo-1 (Figure 5.22) are particularly used formeasuring Ca2+in single cells by imaging or flow cytometry/65... 

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