Fluo-3, fura

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. 

Botana and collaborators also evaluated the effect of ostreocin-D and palytoxin in an excitable cellular model (human neuroblastoma cells). Both toxins caused an important loss of filamentous actin associated to morphological alterations (Fig. 6.7). In neuroblastoma cells, the variation of ion fluxes caused by ostreocin-D and palytoxin was studied by using the membrane potential sensitive probe bis-oxonol and the Ca sensitive dyes fluo-3 and fura red. Ostreocin-D modifies the membrane potential, causing depolarization, although more slight than that produced by palytoxin (Fig. 6.8a). 

Fig. 11. HT cells labeled simultaneously with 5 uM Fluo-3 (detector 1) and 5/iM Fura Red (detector 2).

A powerful new approach is the direct measurement of released Ca + with fluorescent Ca + indicators such as fura-2 or fluo-3, whereby the permeabilized smooth muscle has to be placed into microcuvettes (lino, 1991). Since force is no longer required as an indicator, Ca + release may be measured in the absence of MgATP, which allowed the study of adenine nucleotides on the IP3-induced Ca + release without the interference by Ca + uptake (lino, 1991 Hirose and lino, 1994). Inclusion of ryanodine allows a separate study of the IP3- and caffeine-sensitive stores (Hirose et al., 1993). Experimental protocols have been designed that allow the use of high concentrations of EGTA, thereby preventing Ca -mediated feedback regulation of the Ca2+ release (Hirose and lino, 1994). Direct measurement of Ca + release with indicators becomes even more powerful in combination with... 

Fig. 9.27. Intracellular waves in a guinea-pig cardiac myocyte (Lipp Niggli, 1993). The grey levels represent ratios of the two fluorescent Ca indicators Fluo-3 and Fura-Red. The propagation velocity of the waves is 65 pm/s and the scale bar 20 pm. (Picture kindly provided by Dr E. Niggli.)...

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). 

Calcein, fura-2, BTC, various der. of Calcium Green, fluo-3 AM and fluo-4 AM and some of their dextran conjugates... 

Bailey, S. Macardle, P. J. A flow cytometric comparison of Indo-1 to fluo-3 and Fura Red excited with low power lasers for detecting Ca flux. J. Immunol. Methods 2006, 311, 220-225. 

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