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aCommonly used laser lines and emission detection ranges. ^Depending on specific laser source.

aCommonly used laser lines and emission detection ranges. ^Depending on specific laser source.

cellular processes. Measurement of [Ca2+] in living cells is thus of considerable interest to a broad range of investigators. Until the development of the first practical indicator, quin2, by Tsien et al. (1982), microelectrodes were required to measure [Ca2+] and measurements in small intact cells were impossible. Tsien also introduced the strategy of loading small intact cells with the acetoxymethyl (AM) ester of the dye (Tsien, 1981), an approach now widely used for many dyes. The uncharged AM form of the indicator diffuses freely into the cytoplasm, where it is hydrolyzed by esterases to yield a hydrophilic form which is trapped inside the cell. However, quin2 has several disadvantages that limit its application in flow cyto-metry. In 1985, Grynkiewicz et al. described a new family of highly fluorescent calcium chelators, inaugurating the present popularity of cytometric measurement of [Ca2+]i . One of these dyes, indo-1 ([1-[2-amino-5-[6-carboxy-lindol-2-yl]-phenoxy]-2-[2'-amino-5'-methyl phenoxy]ethane N,N,N',N'-tetraacetic acid]), exhibits large changes in fluorescent emission wavelength upon calcium binding; analysis of the ratio of fluorescence intensities at two wavelengths (~400 and 500 nm) allows calculation of [Ca2+]i independent of variability in cellular size or intracellular dye concentration. The ratiometric approach thus provides this analysis with considerable accuracy. The only significant drawback to the use of indo-1 is the requirement for ultraviolet (UV) excitation. The introduction of a fluorescein-based cal cium-sensitive probe, fluo-3, provided a visibly excited alternative to indo-1 (Minta et al., 1989). This dye exhibits an increase in fluorescence intensity with increasing [Ca2+]i but does not have the advantages of permitting ratiomet-ric determinations; the fluorescence intensity distributions are wider, and calibration is more complicated because the signal is proportional to cell size and dye concentration as well as to [Ca2+]i. Strategies to minimize this problem have been developed, based on the simultaneous use of a second dye to serve as an indicator of the magnitude of dye loading in an individual cell (Rijkers et al, 1990), or the simultaneous use of Fura Red, a dye that exhibits a decrease in fluorescence intensity with increasing [Ca2+]i. The latter combination results in a close simulation of a ratiometric analysis (Novak and Rabinovitch, 1994). Calcium-sensitive dyes useful for flow cytometry are summarized in Table 9.1.4. Protocols for use of indo-1, fluo-3, and fluo-3 combined with Fura Red will be included in a forthcoming supplement.

Measurements of [Ca2+]i have been widely performed as multiparameter analyses, with additional fluorochromes used for the determination of cellular immunophenotype. This allows alterations in [Ca2+]i to be examined in specific immunophenotypic subsets (Rabi-novitch et al., 1986). Fluorescein isothiocy-anate (FITC) and phycoerythrin (PE)-conju-gated antibodies can be used with indo-1, and PE can be used with fluo-3 and the fluo-3/Fura Red combination. Numerous examples of the

Studies of Cell Function

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