Studies on the fluorescence and binding of 8-anilino-1-naphthalene sulfonate by submitochondrial particles

Harris, Robert A.

doi:10.1016/0003-9861(71)90399-7

Cited by 39 publications

(17 citation statements)

References 27 publications

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“…7695) using simple asymptotic analysis (17,41) of dye-cell binding. Two sets of mean fluorescence intensities were computed from the flow cytometric PI histograms and were then plotted in double reciprocal coordinates (see Fig.…”

Section: Calibration Of the Flow Cytometermentioning

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: Calibration Of the Flow Cytometermentioning

confidence: 99%

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

et al. 1995

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“…Even if the data themselves are of good quality, this extrapolation may involve considerable uncertainty. Harris [6] has shown that although such plots may appear linear over a long range, a linear extrapolation to the abscissa may not be justified.…”

Section: Interpretation Of Changes In Ansfmicrosome Fluorescencementioning

confidence: 99%

Fluorescence studies of drug and cation interactions with microsomal membranes

Hawkins

Freedman

1973

FEBS Letters

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“…The fluorescence assay method is normally calibrated by determining the intercept value at infinite protein concentration of a double reciprocal plot of fluorescence yield against protein concentration for a fixed amount of probe (Brocklehurst et al, 1970). In practice, the estimation of the correct intercept value is extremely difficult (Harris, 1971;Layton et al, 1974). The problem is exacerbated by the fact that the values of r/co at low probe concentrations are particularly sensitive to the choice of intercept.…”

Section: Scatchard Binding Plotsmentioning

confidence: 98%

An analysis of the binding of fluorescence probes in mitochondrial systems

Williams¹,

Layton²,

Johnston³

1977

J. Membrain Biol.

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Measurements of the binding of the fluorescent probes 8-anilinonaphthalene-1-sulfonate (ANS) and ethidium ions to whole and disruped mitochondria and submitochondrial particles suggest that the inner mitochondrial membrane is freely permeable to the two probes. Equations relating the binding of permeant probes to the electro-chemical balance across the membrane of vesicular systems are derived and these equations used to analyze Scatchard plots of the binding of the two probes to energized and nonenergized mitochondria and EDTA particles.

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Studies on the fluorescence and binding of 8-anilino-1-naphthalene sulfonate by submitochondrial particles

Cited by 39 publications

References 27 publications

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

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

Fluorescence studies of drug and cation interactions with microsomal membranes

An analysis of the binding of fluorescence probes in mitochondrial systems

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