Flow cytometric analysis of membrane permeability properties influencing intracellular accumulation and efflux of fluorescein

Prosperi, Ennio; Croce, Anna Cleta; Bottiroli, Giovanni; Supino, Rosanna

doi:10.1002/cyto.990070110

Cited by 54 publications

(28 citation statements)

References 14 publications

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“…The cells were double stained using the following procedures: For green fluorescence, 2 . 10' cells/ml were suspended in phosphate-buffered saline (PBS) containing 136 mM NaC1, I 0 mM KH,P04, pH 7.2, and were incubated 1-2 min in the presence of 6 pM carboxyfluorescein-diacetate (Sigma C-8166; from a 1.2 mM stock solution in dimethylsulfoxide) at 37°C (28,29). The CF loading was kept low at about 0.1 fmoV cell, which was determined by using a spectrofluorimeter calibrated for CF.…”

Section: Fluorescent Staining Of Cellsmentioning

confidence: 99%

Electropermeabilization and fluorescent tracer exchange: The role of whole‐cell capacitance

et al. 1995

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Transmembrane crossing of charged fluorescent tracers such as propidium iodide (PI) and carboxyfluorescein (CF) can be used to quantitate membrane permeabilization. Murine myeloma Sp2/0-Ag14 cells were loaded with CF (0.1 fmolkell) before electropulsation (0.5-3.0 kV/cm, 40 ps) in medium containing 25-50 pg/ml PI at 21-23°C. Cytograms of PI vs. CF fluorescence showed three readily distinguishable subpopulations: 1) intact living cells with CF but without PI (these form >95% of the prepulsed population), 2) transiently electropermeabilized but resealed cells showing both CF and low-level PI fluorescence, and 3) permanently permeabilized cells without CF but with very high PI fluorescence. Despite the ready influx of PI, the efflux of CF from transiently permeabilized cells was negligible and was insensitive to pulse parameters; however, electrically killed cells (subpopulation 3) lost all CF fluorescence and probably lost their cytoplasm. This difference in transmembrane passage of the dyes is best explained by binding of intracellular CF to macromolecules (and/or organelles). In isotonic "pulse medium," the membranes resealed after electropulsing with a time constant (73 of about 2 min. In 150 mOsm medium, resealing was faster (typically T~ -0.5 min). The population distribution of PI uptake [coefficient of variation (CV) > 40%] was very broad and could not be accounted for by the radius de-10%). The variability in PI uptake could be explained if the electrical energy of the charged membrane, which depends on the whole-cell capacitance (C,), was taken into account. Evaluation of the C, values with single-cell resolution was based on measurement of the electrical charging time constant of the plasma membrane by electrorotation. Key terms: Plasma membrane, electric breakdown, cytoplasm, fluorescent staining, propidium iodide, dye uptake, electrorotation, carboxyfluorescein, flow cytometry, cell viability Electropermeabilization allows the introduction of foreign substances (e.g., drugs, hormones, proteins, plasmids) into living cells (for reviews, see 27,44,45). This technique has been reported to be the most reliable for studying the effects of normally impermeant molecules on the regulation of cell metabolism. Other permeabilization methods (e.g., the use of bacterial toxins, detergents, hypertonic treatment) can markedly affect cell viability and cause undesirable leakage of the cytoplasm or extensive cell lysis ( 2 5 3 0 ) .Plasma membrane electropermeabilization within statistically representative cell samples can be assessed by measuring membrane crossing of tracer molecules in a flow cytometer. Extremely heterogeneous responses to electropulsing have been found in rather homogeneous (origin, size, morphology) cell samples despite the use of uniform electric fields (parallel plate electrodes) for several cell types (6,9,10,24). Flow cytometry of human red blood cells after electropulsation in the presence of FITCdextran (70 kDa) gave rise to at least three distinct subpopulations: ghosts with negligible upta...

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Section: Fluorescent Staining Of Cellsmentioning

confidence: 99%

Electropermeabilization and fluorescent tracer exchange: The role of whole‐cell capacitance

et al. 1995

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“…Studies with detergents and solvents indicated that the twin probes could be used to ' However, a practical limitation on the use of fluorescein in the present context is imposed by its relatively rapid efflux from a variety of cells with apparently intact cell membranes (6,8,19,22,23). An improved twocolour flow cytometric assay for membrane permeability is described here using a different pair of complementary fluorescent probe molecules.…”

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Multiparametric analysis of cell membrane permeability by two colour flow cytometry with complementary fluorescent probes

Watson

Workman

1990

Cytometry

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We describe an improved twin-probe multiparameter flow cytometric technique to examine cell membrane permeability. Ability to retain preloaded intracellular bis-carboxyethyl carboxy fluorescein (BCECF, green fluorescence) and to exclude extracellular propidium (red fluorescence) is measured, simultaneously with forward and right-angle scatter. This has significant advantages over an earlier method using fluorescein together with ethidium. In addition to the two expected cell populations which were stained green positive, red negative (by convention membrane "intact" and "viable," Region 1) and green negative, red positive ("membrane-damaged" and "non-viable," Region 31, a third population was seen which fluoresced neither green nor red and displayed intermediate light scatter characteristics (Region 2). This was true for each of 9 cell types in vitro. For EMT6 mouse mammary tumour cells held under sub-optimal conditions or treated with membrane-active drugs, progression from Region 1 to Region 2 was observed, followed by further progression from Region 2 to Region 3. Cells eventually accumulated in Region 3. These results suggest that sequential changes in membrane structure lead to increased permeability, first with respect to intracellular BCECF and in turn to extracellular propidium.

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“…The efflux of fluorescein from living cells has been shown to be dependent on membrane integrity and 1 on the metabolic energy supply; indeed, the dye release is affected both by membrane-active agents and mitochondrial poisons (Sengbush et al, 1976;Baisch, 1978;Prosperi et al, 1986). Therefore, the monitoring of the kinetics of fluorescence decrease from anthracycline-treated cells may provide information on alterations induced by the drugs, either on membrane structures or energetic cell metabolism.…”

Section: Discussionmentioning

confidence: 99%

“…In particular, when fluorescein diacetate (FDA) is used as the fluorogenic substrate, the efflux of the fluorescent product (fluorescein) has been shown to be influenced by membrane integrity (Rotman & Papermaster, 1966), inhibited by mitochondrial poisons and modulated by the presence or absence of glucose in the extracellular medium (Prosperi et al, 1986).). Therefore, the monitoring of the kinetics of fluorescence decrease in fluoresceinloaded cells may provide useful information on membrane functions, especially on permeability properties dependent on the metabolic energy supply.…”

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Anthracycline-induced inhibition of membrane permeability functions dependent on metabolic energy

Croce¹,

Prosperi²,

Supino³

et al. 1986

Br J Cancer

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Summary The influence of anthracyclines on membrane permeability functions has been investigated in HeLa cells by monitoring the efflux of fluorescein. Release of the fluorescent dye, dependent on the metabolic energy supply, occurs after the intracellular accumulation and enzymatic hydrolysis of the non-fluorescent substrate fluorescein diacetate (FDA). Flow cytometric evaluation of the efflux kinetics showed that adriamycin (ADR), N-trifluoroacetyladriamycin-14-valerate (AD-32) and daunorubicin (DNR) inhibited the permeability process. The degree of inhibition was dependent, though to different extent, on the intracellular concentration of each drug. An increase in the efflux rate was always observed when the cells were treated with the drugs in the presence of 20 mM glucose. Relationship of these effects with energetic metabolism was supported by the finding that ATP levels were lowered by the drugs and increased by glucose. Evaluation of the cytotoxicity induced by each drug showed that the intracellular amount necessary to inhibit cell survival by 50% was of the same order of magnitude as that which decreases to 50% membrane permeability to fluorescein. These results indicate a correspondence in the concentrations of anthracyclines required for inducing cytotoxicity and for inhibiting membrane permeability functions dependent on the metabolic energy supply.

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Flow cytometric analysis of membrane permeability properties influencing intracellular accumulation and efflux of fluorescein

Cited by 54 publications

References 14 publications

Electropermeabilization and fluorescent tracer exchange: The role of whole‐cell capacitance

Electropermeabilization and fluorescent tracer exchange: The role of whole‐cell capacitance

Multiparametric analysis of cell membrane permeability by two colour flow cytometry with complementary fluorescent probes

Anthracycline-induced inhibition of membrane permeability functions dependent on metabolic energy

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