Evaluation of methods for measuring cellular glutathione content using flow cytometry

Abstract

The currently available flow cytometric stains for cellular glutathione were evaluated, examining the labelling of both human and rodent cell lines under various conditions of concentration, time, and temperature. Procedures were used that depleted glutathione (GSH) while having a minimal effect on other cellular sulphydryls in order to estimate linearity and the extent of background staining. As previously reported, monochlorobimane was highly specific for GSH in rodent cells but failed to label human cells adequately because of its low affinity for human glutathione S-transferases. Higher concentrations of monochlorobimane achieved more complete labelling of the human cellular GSH pool but gave increased background fluorescence due to non-GSH binding. The analogue monobromobimane, which binds nonenzymatically to sulphydryls, reacted more readily with GSH than with protein sulphydryls and, provided that stain concentration and incubation time were controlled, gave reproducible staining of human cells with -20% of total fluorescence due to background staining. Of the currently available stains for measuring GSH in human cells, monobromobimane is the agent of choice. Mercury orange also binds more readily to GSH than to protein, giving a degree of specificity, and it has the additional advantage of being excited at 488 nm. However, the reproducibility of staining with mercury orange was less consistent than that using monobromobimane, and a higher background fluorescence was seen. Two additional stains, o-phthaldialdehyde and chloromethyl fluorescein, could also be used to label cellular GSH, but both gave an unacceptably high level of background staining. It is recommended that flow cytometric GSH assays should routinely include a sample of cells that have been depleted of GSH in order to determine the extent of background labelling. 0 1994 Wiley-Liss, Inc.

Busulfan-induced senescence is dependent on ROS production upstream of the MAPK pathway

“…After overnight growth, they were treated with BU for various durations and then incubated with monochlorobimane (mBCI, 40μM) (Invitrogen) in a staining solution (5mM glucose, 1mM CaCl 2 , 0.5mM MgSO 4 , 5 mg/ ml BSA) for 30 min at 37 °C in the dark. Although mBCI is a nonfluorescent probe, it forms a stable fluorescent adduct with GSH in a reaction catalyzed by the GSH S-transferases [25]. The mean fluorescent intensity of the fluorescent GSH-bimane adduct was measured using a Spectra Max Gemini XS fluorescent plate reader (Molecular Devices Corp., Sunnyvale, CA) at λ ex =380 nm and λ em =460 nm to detect GSH [25].…”

“…Although mBCI is a nonfluorescent probe, it forms a stable fluorescent adduct with GSH in a reaction catalyzed by the GSH S-transferases [25]. The mean fluorescent intensity of the fluorescent GSH-bimane adduct was measured using a Spectra Max Gemini XS fluorescent plate reader (Molecular Devices Corp., Sunnyvale, CA) at λ ex =380 nm and λ em =460 nm to detect GSH [25].…”

“…Therefore, we hypothesized that BU may activate Erk and p38 by inducing oxidative stress. To test this hypothesis, we first examined the effect of BU on the intracellular levels of GSH using mBCI which readily enters cells to form a fluorescent GSH-bimane adduct that can be measured fluorometrically [25]. As shown in Fig.…”

In vitro circumvention of cisplatin resistance by the novel sterically hindered platinum complex AMD473

“…Cells were then instantly exposed to platinum agents for 2 h followed by incubation in drugfree growth medium for a further 94 h. IC50 values for cisplatin and AMD473 were then determined as in the above SRB assay. Approximately 1 x 106 A2780 ovarian carcinoma cells seeded in T25 tissue culture flasks were treated with GSH ester in an identical way and measured for relative intracellular GSH content by flow cytometry using monobromobimane fluorescence as an indicator of relative GSH (Hedley and Chow, 1994 (Lowry et al, 1951). The remainder of the sonicated cells were then analysed by flameless atomic absorption spectrophotometry (FAAS) for platinum content using Perkin Elmer models HGA700 and 1 IOOB.…”

CD4+ lymphocytes from HIV-infected patients display impaired CD45-associated tyrosine phosphatase activity which is enhanced by anti-oxidants

“…Low molecular weight thiol (LMT) levels were estimated by flow cytometry using the 5-chloromethyl fluorescein diacetate (CMF) probe [17]. This non-fluorescent probe has been demonstrated to form fluorescent adducts with intracellular low molecular thiols, of which GSH is by far the most abundant.…”