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monocytes, in which DCFH-DA hydrolysis is incomplete (Robinson et al., 1988). The sensitivity and response time of oxidative burst detection by DHR 123 and DCFH-DA are impaired by their location in the cytoplasm rather than at the site of oxidative activity in the phagocytic vacuole. In addition, false positive responses can be generated in unstimulated cells because of intercellular diffusion of H2O2 (Henderson and Chappell, 1993). Coupling DCFH to an immune complex overcomes these problems, producing a probe targeted to the phagocytic vacuole via Fc receptor-mediated internalization (Ryan et al., 1990).

The response mechanism of dihydro-ethidium (also frequently referred to as hydro-ethidine) to intracellular oxidants is considerably different from that of DCFH-DA (Fig. 4.4.6B). Unlike DHR123 or DCFH-DA, dihy-droethidium can be oxidized directly by superoxide anion (O2-), which is generated earlier in the oxidative burst reaction sequence than H2O2 (Rothe and Valet, 1990). The spectral characteristics of DNA-bound ethidium (absorption and emission maxima at 518 nm and 605 nm respectively) are suitable for simultaneous excitation and detection in combination with DCFH-DA. This procedure provides better discrimination between functionally distinct neu-trophil populations than does single-probe analysis (Rothe and Valet, 1990; Robinson et al., 1994b). Dihydroethidium and dihydrorho-damine 6G (a close analog of DHR123) exhibit increased accumulation in epithelial cells expressing the cystic fibrosis transmembrane conductance regulator, and can therefore be used to detect functional activation of this cAMP-dependent chloride channel (Wersto et al., 1996).

Probes for Intracellular Thiols

The tripeptide glutathione (GSH; y-Glu-Cys-Gly) is the most abundant nonprotein thiol in eukaryotic cells. Its primary role is to provide the reducing capacity required to prevent damage to DNA by free radicals and other oxidants. GSH also participates in the removal of xeno-biotics, thereby contributing to the develop-

ment of multidrug resistance. The primary problems encountered in flow cytometric determination of GSH are obtaining a stoichiometric fluorescence response to the prevailing high intracellular GSH concentrations (typically >2 mM) and specificity for GSH versus other thiols (Hedley and Chow, 1994a,b).

Monochlorobimane (mBCl) and monobro-mobimane (mBBr) are currently the most widely used probes for intracellular GSH. Both are freely membrane permeant and initially nonfluorescent; conjugation to GSH yields a fluorescent thioether with fluorescence excitation and emission maxima at ~390 and 480 nm, respectively (Fig. 4.4.7A). Because the thiol reactivity of mBCl is low, coupling to GSH only occurs to a significant extent via the action of glutathione-S-transferase (GST). Coupling of the more reactive mBBr occurs spontaneously. Although enzymatic mediation of mBCl coupling provides high GSH specificity, the probe is unfortunately a poor substrate for many GST isoenzymes, leading to underestimation of GSH levels (Ublacker et al., 1991; Hedley and Chow, 1994a). For this reason, mBBr is generally preferred over mBCl (Hedley and Chow, 1994a,b). Extrusion of the fluorescent conjugate by organic ion transporters may also contribute to underestimation of GSH (Poot et al., 1996a).

Cell-permeant 5-chloromethylfluorescein diacetate (CMFDA) is converted to a fluorescent product by the action of intracellular esterases. The cleavage product (5-chloro-methylfluorescein) can then couple to GSH via the same enzymatic process as mBCl. The significant difference in the case of CMFDA is that GSH coupling produces no change in fluorescence (Fig. 4.4.7B). The principal advantage of CMFDA over mBBr and mBCl is that its absorption maximum is well matched to the 488-nm argon-ion laser line. CMFDA is less GSH specific than the mBBr and mBCl according to published comparisons (Poot et al., 1991; Barhoumi et al., 1993; Hedley and Chow, 1994a). Discrepancies appear to arise from the fluorescence of unconjugated 5-chloromethyl-fluorescein rather than from conjugation to thiols other than GSH (Hedley and Chow, 1994a). Nevertheless, GSH levels measured using CMFDA have correlated well with those determined using other methods in several instances (Barhoumi et al., 1993; Coates and Tripp, 1995).

Coupling of CMFDA to GSH and other intracellular thiols provides a basis for long-term retention of the probe in the cells, except in cases where retention is compromised by active extrusion of the fluorescent conjugate (see above). Labeling of cell populations with well-retained fluorescent tracers allows functional properties expressed at the multicellular level, such as adhesion, fusion, and gap junction communication, to be detected and quantitated.

A general experimental strategy for these applications is to label two cell populations with probes that have contrasting emission spectra; cell-cell interactions result in probe transfer and the subsequent detection of a third, dual-labeled cell population. For example, CMFDA has been used in combination with an analogous rhodamine derivative (5-[and-6]-[{(4-chloromethyl)benzoyl}amino] tetraethyl-rhodamine) to quantitatively detect hybrid cells produced by electrofusion (Jaroszeski et al., 1994).

Other probes resistant to spontaneous intercellular transfer for these applications include amine-reactive carboxyfluorescein diacetate succinimidyl ester (CFDA SE), thiol-reactive 4,5,6,7-tetrafluorofluorescein diacetate (TFFDA), calcein AM, and lipophilic carbocyanines (see below). Unlike CMFDA (Fig. 4.4.7B), TFFDA is only thiol reactive prior to esterase cleavage; the fluorescent product 4,5,6,7-tetrafluorofluorescein is not reactive (Gee et al., 1996). CFDA SE is widely used to label cells for in vivo migration studies (Weston and Parish, 1990). Because the label distributes equally among cell progeny, successive generations of dividing cells are marked by halving of cellular fluorescence (Lyons and Parish, 1994).

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