gramicidin-a and bis(1-3-dibutylbarbiturate)trimethine-oxonol

Quantitative determinations of the cell membrane potential of lymphocytes (Wilson et al., J Cell Physiol 1985;125:72-81) and thymocytes (Krasznai et al., J Photochem Photobiol B 1995;28:93-99) using the anionic dye DiBAC4 (3) proved that dye depletion in the extracellular medium as a result of cellular uptake can be negligible over a wide range of cell densities. In contrast, most flow cytometric studies have not verified this condition but rather assumed it from the start. Consequently, the initially prepared extracellular dye concentration has usually been used for the calculation of the Nernst potential of the dye. In this study, however, external dye depletion could be observed in both large IGR-1 and small LCL-HO cells under experimental conditions, which have often been applied routinely in spectrofluorimetry and flow cytometry. The maximum cell density at which dye depletion could be virtually avoided was dependent on cell size and membrane potential and definitely needed to be taken into account to ensure reliable results. In addition, accepted calibration procedures based on the partition of sodium and potassium (Goldman-Hodgkin-Katz equation) or potassium alone (Nernst equation) were performed by flow cytometry on cell suspensions with an appropriately low cell density. The observed extensive lack of concordance between the correspondingly calculated membrane potential and the equilibrium potential of DiBAC4 (3) revealed that these methods require the additional measurement of cation parameters (membrane permeability and/or intracellular concentration). In contrast, due to the linear relation between fluorescence and low DiBAC4 (3) concentrations, the Nernst potential of the dye for totally depolarized cells can be reliably used for calibration with an essentially lower effort and expense.

Topics: Algorithms; Barbiturates; Calcium Ionophores; Calibration; Cell Line; Cell Membrane Permeability; Flow Cytometry; Fluorescent Dyes; Gramicidin; Humans; Ionomycin; Isoxazoles; Membrane Potentials; Models, Biological; Staining and Labeling

In this study, the authors compared and evaluated 4 membrane potential probes in the same cellular assay: the oxonol dye DiBAC(4)(3), the FLIPR membrane potential (FMP) dye (Molecular Devices), and 2 novel fluorescence resonance energy transfer (FRET) dye systems from PanVera [CC2-DMPE/DiSBAC(2)(3)] and Axiom [DiSBAC(1)(3)/DiSBAC(1)(5)]. The kinetic parameters of each membrane probe were investigated in RBL-2H3 cells expressing an endogenous inward rectifier potassium channel (IRK1). The FMP dye presented the highest signal over background ratio whereas the FRET dyes from PanVera gave the fastest response. The determination of IC(50) values for 8 different channel modulators indicated a good correlation between the 4 membrane probe systems. The compound-dye interaction was evaluated in the presence of compounds at 10 muM and clearly indicated no effect on the FMP or the PanVera donor dye, whereas some major interference with the oxonol probes was observed. Using a cell permeabilization assay in the presence of gramicidin, the authors concluded that the FRET dyes from PanVera and the FMP dye are unable to measure the gramicidin-induced cell membrane hyperpolarizations. The 4 dye systems were investigated under high-throughput screening (HTS) conditions, and their respective Z' parameter was determined. The characteristics of each dye system and its potential use in HTS assays is discussed.

Topics: Animals; Barbiturates; Cell Membrane Permeability; Cells, Cultured; CHO Cells; Cricetinae; Drug Evaluation, Preclinical; Drug Interactions; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Gramicidin; Inhibitory Concentration 50; Ion Channels; Isoxazoles; Kinetics; Membrane Potentials; Molecular Biology; Potassium Channels, Inwardly Rectifying; Rats; Thiobarbiturates

Flow cytometry using the anionic membrane potential-sensitive fluorescent probe, bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC4(3)), enabled assessment of antibiotic-induced membrane perturbation in five clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and two antibiotic-sensitive reference strains, NCTC 6571 and 8325-4, after establishment of steady-state growth in liquid cultures inoculated from single colonies. Flow cytometric indications of the enhanced DiBAC4(3) uptake after treatment with vancomycin at 0.1, 1, 4 and 10 x MIC showed excellent comparison with viability losses quantified as cfu on solid agar in MRSA isolate QC. The antibiotic susceptibility patterns to benzylpenicillin, methicillin and vancomycin for all isolates used in this study could be determined in 2-4 h from an overnight plate culture. This technique thus provides a rapid and reproducible antibiotic sensitivity test which may be applicable in routine clinical practice.

Topics: Anti-Bacterial Agents; Barbiturates; Flow Cytometry; Gramicidin; Hot Temperature; Isoxazoles; Methicillin; Methicillin Resistance; Microbial Sensitivity Tests; Penicillin G; Reproducibility of Results; Staphylococcus aureus; Vancomycin

1. The present study was designed to test if microvascular coronary endothelial cells express ATP-sensitive K+ channels (KATP channels). We performed microfluorometric measurements of the membrane potential of freshly isolated guinea-pig coronary capillaries equilibrated with the voltage-sensitive dye bis-oxonol (bis-[1,3-dibutylbarbituric acid] trimethineoxonol, [DiBAC4(3)]). 2. The resting membrane potential of capillaries in physiological salt solution was -46 +/- 4.2 mV (n = 8) at room temperature (22 degrees C) as determined after calibration of the fluorescence using the Na(+)-K+ ionophore gramicidin in the presence of different K+ concentrations. Spontaneous membrane potential fluctuations of 10-20 mV amplitude were often observed. 3. A reversible, sustained hyperpolarization to a new membrane potential close to the K+ equilibrium potential (EK) could be induced by application of the K+ channel openers HOE 234 (100 nM to 1 microM), diazoxide (10 PM to 100 nM) or pinacidil (100 nM). Subsequent addition of glibenclamide (200 nM to 2 microM) reversed this hyperpolarization. 4. A glibenclamide-sensitive hyperpolarization of coronary capillaries to values near EK was also observed upon omission of D-glucose (10 mM) from the superfusing solution or by substituting L-glucose for D-glucose. Maximum hyperpolarization was reached in less than 10 min. 5. Our results suggest that microvascular coronary endothelial cells express KATP channels which may be activated during hypoglycaemia.

Topics: Adenosine Triphosphate; Animals; Barbiturates; Capillaries; Chromans; Coronary Circulation; Diazoxide; Fluorescent Dyes; Glucose; Glyburide; Gramicidin; Guanidines; Guinea Pigs; In Vitro Techniques; Isoxazoles; Membrane Potentials; Muscle, Smooth, Vascular; Osmolar Concentration; Pinacidil; Potassium; Potassium Channels; Pyrrolidines; Sodium Chloride; Vasodilator Agents

CD3 mAb induced calcium movements are unaffected by hyperpolarization of the membrane in Jurkat T cells treated with valinomycin. By contrast, the CD3 induced Ca2+ influx was impaired by depolarization of the membrane with either gramicidin or by equimolar substitution of KCl for NaCl in the medium. In depolarized cells, the synthesis of phosphatidylserine was strongly diminished as a result of impaired transport of the [3H]serine substrate. In depolarized cells, the CD3-induced release of Ca2+ from intracellular stores (endoplasmic reticulum) was unaffected. Emptying of the Ca2+ stores by CD3 was shown by the lack of effect of additional treatment of the cells with the Ca2+ ionophore, ionomycin. The empty status of the calcium stores was also confirmed by measurements of phosphatyidylserine synthesis through the Ca2+ -dependent base exchange enzyme system that was found to be significantly decreased despite the low amount synthesized in the presence of a defective [3H]serine transport in depolarized cells.

Topics: Antibodies, Monoclonal; Barbiturates; Calcium; CD3 Complex; Fluorescent Dyes; Gramicidin; Humans; Indoles; Ionomycin; Ionophores; Isoxazoles; Jurkat Cells; Membrane Potentials; Phosphatidylserines; T-Lymphocytes; Valinomycin

Various dyes were assessed for their ability to discriminate between viable and non-viable bacteria. Two methods of killing were employed: by heat treatment or by gramicidin treatment. Staining was carried out in two ways; by staining directly in the medium or by washing cells prior to staining in buffer. Carbocyanine and rhodamine 123 dyes only exhibited small changes in fluorescence between viable and non-viable populations of bacteria. Both oxonol dye (bis 1,3-dibutylbarbituric acid trimethine oxonol) and calcafluor white proved much more useful.

Topics: Bacterial Physiological Phenomena; Barbiturates; Carbocyanines; Colony Count, Microbial; Escherichia coli; Flow Cytometry; Fluorescent Dyes; Gramicidin; Heating; Isoxazoles; Membrane Potentials; Organic Chemicals; Rhodamine 123; Rhodamines; Salmonella typhimurium; Staining and Labeling; Staphylococcus aureus