Octadecylrhodamine B Research Articles

Flow cytometry-based FRET to monitor β2 integrins activation

Abstract β2 integrins is very important for leukocyte arrest, rolling and transmigration. Neutrophils mainly express two β2 integrins, αLβ2 and αMβ2. Neutrophil arrest is triggered by rapid conformational change of β2 integrins from bent and low affinity to open and high affinity. Recent studies have reported a new conformation of β2 integrins bent high affinity (PMID: 27578049, PMID: 30605669). This conformation is very difficult to monitor. By dynamic molecular simulation, we found that bent high affinity conformation of integrin is close enough to membrane, less than 10nm. Förster resonance energy transfer (FRET) is a powerful technology to interrogate close association between two fluorescent molecules. Importantly, FRET requires a distance of 10nm or less. Using real-time flow cytometry-based FRET, we can not only detect bent high affinity conformation but also the dynamic conformational change of β2 integrins along time. Interestingly, the FRET signal was detected between bent high affinity β2 integrins and cell membrane using conformational antibody mAb24 and membrane dye octadecyl rhodamine B (ORB). We developed an easy and efficient way to monitor the bent high affinity conformation of β2 integrins.

Detergent Induction of HEK 293A Cell Membrane Permeability Measured under Quiescent and Superfusion Conditions Using Whole Cell Patch Clamp

Detergents have several biological applications but present cytotoxicity concerns, since they can solubilize cell membranes. Using the IonFlux 16, an ensemble whole cell planar patch clamp, we observed that anionic sodium dodecyl sulfate (SDS), cationic cetyltrimethylammonium bromide (CTAB), and cationic, fluorescent octadecyl rhodamine B (ORB) increased the membrane permeability of cells substantially within a second of exposure, under superfusion conditions. Increased permeability was irreversible for 15 min. At subsolubilizing detergent concentrations, patched cells showed increased membrane currents that reached a steady state and were intact when imaged using fluorescence microscopy. SDS solubilized cells at concentrations of 2 mM (2× CMC), while CTAB did not solubilize cells even at concentrations of 10 mM (1000× CMC). The relative activity for plasma membrane current induction was 1:20:14 for SDS, CTAB, and ORB, respectively. Under quiescent conditions, the relative ratio of lipid to detergent in cell membranes at the onset of membrane permeability was 1:7:5 for SDS, CTAB, and ORB, respectively. The partition constants (K) for SDS, CTAB, and ORB were 23000, 55000, and 39000 M–1, respectively. Combining the whole cell patch clamp data and XTT viability data, SDS ≤ 0.2 mM and CTAB and ORB ≤ 1 mM induced cell membrane permeability without causing acute toxicity.

Vesicular transfer of membrane components to bovine epididymal spermatozoa

Epididymosomes (apocrine secreted epididymal vesicles) are assumed to play a crucial role in sperm maturation. Our aim has been to analyze the fusogenic properties of bovine epididymosomes and their involvement in the transfer of membrane components (lipids, proteins, plasma membrane Ca(2+)-ATPase 4 [PMCA4]) into bovine sperm. The fusogenic properties of epididymosomes with spermatozoa were investigated in vitro by using octadecyl rhodamine-B (R18)-labeled epididymosomes. Spermatozoa isolated from the epididymal caput showed a higher fusion rate than those taken from the cauda. The fusion rate was dependent on pH and time. Furthermore, the lipid and protein content in spermatozoa changed during epididymal transit and after in vitro fusion with epididymosomes. Following the in vitro fusion of caput spermatozoa with epididymosomes, the cholesterol/total phospholipid ratio of the sperm plasma membrane decreased. The effect was comparable with the cholesterol/total phospholipid ratio of native cauda spermatozoa. Co-incubation experiments of spermatozoa with biotinylated epididymosomes additionally revealed that proteins were transferred from epididymosomes to sperm. To examine the potential transfer of epididymis-derived PMCA4 to spermatozoa, immunofluorescence analysis and Ca(2+)-ATPase activity assays were performed. In caput spermatozoa, the PMCA4 fluorescence signal was slightly raised and Ca(2+)-ATPase activity increased after in vitro fusion. Thus, our experiments indicate significant changes in the lipid and protein composition of epididymal sperm following interaction with epididymosomes. Moreover, our results substantiate the presumption that PMCA4 is transferred to spermatozoa via epididymosomes.

Fluorescence Anisotropy of Hydrophobic Probes in Poly(N-decylacrylamide)-block-poly(N,N-diethylacrylamide) Block Copolymer Aqueous Solutions: Evidence of Premicellar Aggregates

Fluorescent probes, coumarin 153 (C153) and octadecylrhodamine B (ORB), were used to study the self-assembly in water of poly(N-decylacrylamide)-block-poly(N,N-diethylacrylamide), (PDcA(11)-block-PDEA(295); M(n) = 40 300 g mol(-1); M(w)/M(n) = 1.01). From the variation of both the fluorescence intensity and the solvatochromic shifts of C153 with polymer concentration, the critical micelle concentration (CMC) was determined as 1.8 +/- 0.1 microM. On the other hand, steady-state anisotropy measurements showed the presence of premicellar aggregates below the CMC. Time-resolved fluorescence anisotropy evidenced that ORB is located in the premicellar aggregates and the micelle core, while C153 is partitioned between the aggregates and the water phase. The micelle core contains both semicrystalline and amorphous regions. In the semicrystalline regions the probes cannot rotate, while in the amorphous regions the rotational correlation times correlate well with the hydrodynamic volume of the probes. The amorphous region of the micelle core is relatively fluid, reflecting the large free-volume accessible to the probes.

Electronic Excitation Energy Ttransfer of Prodan-ORB and Laurdan-ORB Systems in Model Phospholipid Membranes

Electronic energy transfer between prodan (6-propionyl-2-dimethylaminonaphthalene) and laurdan (6-dodecanoyl-2-dimethylaminonaphthalene) acting as donors and ORB (octadecyl rhodamine B) acting as acceptor in a model system of membranes has been studied by a steady-state fluorescence quenching analysis. Factors determining the energy transfer rate were considered with special attention to the contribution from the spectral heterogeneity of the donor molecules. By analyzing experimental data within the framework of two theoretical models (developed by Stern, Volmer, and Lehrer) of the energy transfer in two-dimensional systems, the limits of the range of possible prodan and laurdan positions with respect to a lipid bilayer have been estimated.

Direct Fusion between Poly(ethylene oxide)-lipid Modified Liposomes and Murine Mitotic B16 Melanoma Cells

The interactions between the poly(ethylene oxide)-bearing lipid (PEO-lipid) were investigated with the average number of ethylene oxide units of 5, 15 and 32-reconstituted egg PC liposomes and murine mitotic B16 melanoma cells. Water-soluble FITC-dextran (20kDa) and lipophilic octadecyl rhodamine B (OD-RhoB) were encapsulated in liposomes to study the interaction modes with these cells by fluorescence microscopic techniques. Both fluorescent probes loaded in the PEO-lipid (n 32, 20mol%)-reconstituted liposome were specifically transferred to the mitotic cells. This process was not inhibited at 4°C or after the treatment of endocytosis inhibitor cytochalasin B or D. Confocal fluorescence microscopic observation of the cells treated with the liposome at 4°C revealed that FITC-dextran and OD-RhoB were transferred to the cytosol and the plasma membrane, respectively. In addition, when the mitotic cells were treated with the PEO-lipid (n 32, 20mol%)-reconstituted liposome encapsulated diphtheria toxin fragment A (DTA), approximately 30% of the cells were killed by the DTA-dependent cytotoxicity. These data indicate that the PEO-lipid (n 32, 20mol%)-reconstituted liposome directly fused with the plasma membrane of the murine mitotic B16 melanoma cells.

Photoinduced electron transfer and geminate recombination in the group head region of micelles

A pump-probe spectroscopic study of photoinduced forward electron transfer and geminate recombination between donors and acceptors located in the head group regions of micelles is presented. The hole donor is octadecyl-rhodamine B (ODRB) and the hole acceptor is N,N-dimethyl-aniline (DMA). The experiments are conducted as a function of the DMA concentration in the dodecyltrimethylammonium bromide and tetradecyltrimethylammonium bromide micelles. In spite of the fact that the absorptions of both the ODRB radical and ground state bleach spectrally overlap with the ODRB excited state absorption, a procedure that makes it possible to determine the geminate recombination dynamics is presented. These experiments are the first to measure the dynamics of geminate recombination in micelles, and the experiments have two orders of magnitude better time resolution than previous studies of forward transfer. The experimental data are compared to statistical mechanics theoretical calculations of both the forward transfer and the geminate recombination. The theory includes important aspects of the topology of the micelle and the diffusion of the donor-acceptors in the micelle head group region. A semiquantitative but nonquantitative agreement between theory and experiments is achieved.

Phase transition affects energy transfer efficiency in phospholipid vesicles

The fluorescence quenching of 6-propionyl-2-dimethylaminonaphtalene (PRODAN) and 6-dodecanoyl-2-dimethylaminonaphtalene (LAURDAN) by octadecyl rhodamine B (ORB) in a model system of small unilamellar vesicles (SUV) of dipalmitoylphosphatidyl-choline (DPPC) was investigated. Non-linear Stern–Volmer behaviour was observed in both systems in the gel phase (25 °C) and in the fluid phase (50 °C), resulting from association processes and from static quenching. The relative quenching efficiencies of both dyes depend on the phase state of the bilayer and indicate a deeper incorporation of PRODAN and LAURDAN into the membrane in its fluid phase than in its gel phase.

Fluorescence Correlation Spectroscopy Using Octadecylrhodamine B as a Specific Micelle-Binding Fluorescent Tag; Light Scattering and Tapping Mode Atomic Force Microscopy Studies of Amphiphilic Water-Soluble Block Copolymer Micelles,

Amphiphilic multimolecular block copolymer micelles of polystyrene-block-poly(methacrylic acid) and polystyrene-block-poly(ethylene oxide) were investigated by fluorescence correlation spectroscopy (FCS) in aqueous media using octadecylrhodamine B (ORB) as a micelle-specific probe, together with UV−visible absorption and other fluorescence techniques (steady-state spectroscopy and depolarization and time-resolved fluorescence decay measurements). The aim of the study was to show that the fluorescence techniques alone provide sufficient information on the micellar system, allowing, thus, for the fully conclusive study. Other techniques, such as static and quasi-elastic light scattering and atomic force microscopy were used just for comparison to prove the reliability of the FCS results and estimate their accuracy. The binding kinetics of ORB to micelles in aqueous solutions accompanied by association-dependent changes in UV−vis and fluorescence spectra and the binding equilibrium were also studied because ...

Octadecylrhodamine B as a Specific Micelle-Binding Fluorescent Tag for Fluorescence Correlation Spectroscopy Studies of Amphiphilic Water-Soluble Block Copolymer Micelles. Spectroscopic Behavior in Aqueous Media

Fluorescence behavior of octadecylrhodamine B (ORB) and the association-dependent changes in UV-VIS absorption and fluorescence spectra in aqueous solutions were studied by UV-VIS absorption spectroscopy and steady-state and time-resolved fluorometry. Spectral changes are interpreted in terms of the hydrophobically driven formation of H- and J-dimers and higher aggregates. The knowledge gained serves as a basis for the interpretation of results of the fluorescence correlation spectroscopy study of polymeric micelles labeled with ORB.

Light Scattering, Atomic Force Microscopy and Fluorescence Correlation Spectroscopy Studies of Polystyrene-block-poly(2-vinylpyridine)-block-poly(ethylene oxide) Micelles

Polymeric nanoparticles formed by triblock copolymer polystyrene-block-poly(2-vinylpyridine)-block-poly(ethylene oxide), PS-PVP-PEO, in aqueous media were studied by a combination of fluorescence correlation spectroscopy with other fluorescence techniques, light scattering and atomic force microscopy. The studied polymeric nanoparticles exist in the form of (i) core/shell micelles in acid solution at pH lower than 4.8 and (ii) three-layer onion micelles at higher pH. Since water is a very strong precipitant for PS, both types of micelles have kinetically frozen spherical PS cores. The cores of micelles in acid media are surrounded by soluble shells formed by partly protonated PVP and PEO, while the cores of micelles in alkaline media are surrounded by compact insoluble layers of deprotonated PVP and soluble PEO shells. The micellization behavior of PS-PVP-PEO micelles is accompanied by secondary aggregation of micelles, which is provoked by stirring, shaking and also by filtration of micellar solutions. Therefore fluorescence correlation spectroscopy (FCS), which, in contrast to light scattering techniques, does not require filtration, was used as the main experimental technique for the characterization of non-aggregated micelles. The binding of a fluorescence probe, octadecylrhodamine B (ORB), to polymeric micelles, was studied before the FCS study of micelles.

Photoinduced Intermolecular Electron Transfer in Micelles: Dielectric and Structural Properties of Micelle Headgroup Regions

Photoinduced intermolecular (donor/acceptor) electron transfer is studied both experimentally and theoretically for donors and acceptors located in the headgroup region of micelles. Fluorescence up-conversion and fluorescence yield measurements were performed to characterize photoinduced electron transfer from N,Ndimethylaniline (DMA) and N,N-dimethyl-1-naphthylamine (DMNA) to octadecylrhodamine B (ODRB) in three types of aqueous micelle solutions: dodecyl-, tetradecyl-, and cetyltrimethylammonium bromide (DTAB, TTAB, and CTAB, respectively). The data were analyzed with a detailed theory that assumes a Marcus distance-dependent rate constant. Because DMA, DMNA, and ODRB reside in the headgroup region of the micelles, the theory includes diffusion of the molecules in this region of the micelles. The micelles are modeled as a spherical core of low dielectric constant surrounded by a spherical shell headgroup region with intermediate dielectric properties, which in turn is surrounded by water. An analytical theory, which accounts for geometrical and dielectric properties of the three-region micelle environment, is used to calculate the solvent reorganization energy and free energy of transfer. To fit the data, the three-region dielectric model is necessary, and the dielectric constant of the micelle headgroup region of each micelle can be approximately determined. In addition, including local structure is required to fit the data, yielding some information about molecular organization in the headgroup region.

Solvent Reorganization Energy and Free Energy Change for Donor/Acceptor Electron Transfer at Micelle Surfaces: Theory and Experiment

Theories are presented for calculating the solvent reorganization energy and the free energy change which occur in photoinduced donor/acceptor electron transfer at the surface of micelles. The theories are based on the Marcus theory for spherical reactants in a dielectric continuum. The micelle is modeled with regions of differing dielectric properties, representing the micelle core, the headgroup region, and the surrounding water. The free energy change accompanying electron transfer can be calculated from redox measurements made in bulk liquids. The theories are applied to previously published photoinduced intermolecular electron-transfer data between octadecylrhodamine B (ODRB) and N,N-dimethylaniline (DMA) molecules.1 The ODRB and DMA molecules are located in the surface region of three different types of surfactant micelles: dodecyl-, tetradecyl-, and cetyl-trimethylammonium bromide (DTAB, TTAB, and CTAB, respectively). The data show an increased rate of electron transfer with increasing micelle rad...

Photoinduced Electron Transfer on the Surfaces of Micelles

Photoinduced electron transfer between N,N-dimethylaniline (DMA) and octadecylrhodamine B (ODRB) is studied on the surfaces of three alkyltrimethylammonium bromide micelles: dodecyl- (DTAB), tetradecyl- (TTAB), and hexadecyltrimethylammonium bromide (CTAB). The DMA and ODRB molecules are localized at the micelle surface. Time-resolved fluorescence and fluorescence yield data are presented and analyzed with the theoretical methods of ref 1. Lateral diffusion of the molecules over the micelle surfaces is included. Although the three micelles are structurally similar, pronounced differences in the electron-transfer kinetics are observed, with the overall amount of electron transfer increasing with alkyl chain length for the same DMA surface packing fraction. This result is attributed to differences in the solvent reorganization energy, possibly due to varying extents of water penetration into the headgroup regions of the three micelles. As the surfactant chain length increases, the solvent reorganization energy is reduced, resulting in faster electron transfer.

FLUORESCENCE STUDIES ON THE ADSORPTION OF OCTADECYLRHODAMINE B ONTO A LATEX SURFACE

Abstract— The interaction of octadecylrhodamine B (ORB), a lipid‐conjugated dye, with polystyrene latex in aqueous dispersions has been investigated by fluorescence techniques. The dependence of the fluorescence intensity of ORB on the latex concentration indicates that the dye is effectively adsorbed onto the latex surface from the aqueous phase and that deaggregation to fluorescent monomers from the nonfluorescent multimers takes place cooperatively when the surface coverage of the dye is reduced to a critical value. The fluorescence lifetime increases on going from a homogeneous methanolic solution to the latex dispersion, showing that the mobility of the probe is suppressed on the latex surface. From the results of polarization measurements, it is proposed that ORB molecules are adsorbed on the latex surface by a side‐on geometry. The influence of a nonionic surfactant, triton X‐100, on the fluorescence intensities of ORB and rhodamine B in the latex dispersion is also investigated.

PHOTOLUMINESCENT PROPERTIES OF OCTADECYLRHODAMINE B IN MICELLES OF LOW-MOLECULAR-WEIGHT DETERGENTS AND WATER-SOLUBLE TRIBLOCK COPOLYMERS

The fluorescence intensity, lifetime and degree of polarization of octadecylrhodamine B (ORB) have been measured in order to examine the usefulness of this molecule as a probe of micelle properties for low-molecular-weight detergents and water-soluble triblock copolymers. The surfactants examined are hexadecyltrimethylammonium chloride (HTAC), Triton X-100 (TX-100), sodium dode-cylsulfate (SDS), sodium tetradecylsulfate (STS), and Pluronic L64 (ethylene oxide [EO]13 propylene oxide30 EO13, L64). The fluorescence intensity and degree of polarization of ORB show drastic increases at the critical micelle concentrations (CMC) of HTAC, TX-100 and L64, indicating that ORB is cooperatively incorporated into the micelles upon micellization. This feature demonstrates the validity of ORB as a probe for detecting micelle formation of these surfactants. However, in the case of SDS and STS, the fluorescence intensity starts to rise at concentrations far below the CMC, and the degree of polarization does not show significant changes at the CMC. The details of the interactions between ORB and the anionic surfactants have been unclear. These facts imply that some caution is needed for the applications of ORB to the systems containing anionic surfactants. The local viscosity of L64 micelles has been determined by polarization and lifetime measurements. The structure of the block copolymer micelles and the locations of the probe in the micelles are discussed in terms of the viscosity data.

PHOTOLUMINESCENT PROPERTIES OF OCTADECYLRHODAMINE B IN WATER, IN ALCOHOLS AND IN MIXED WATER‐ALCOHOL SOLUTIONS

Abstract Fluorescence, excitation and absorption spectra of octadecylrhodamine B (ORB) have been observed in solutions of water, methanol, ethanol and other simple alcohols. A remarkable increase in the ORB fluorescence intensity occurs when the probe is transferred from water to the alcohols. This effect is ascribed to the release of ORB fluorescent monomers from nonfluorescent multimers, which exist predominantly in water. The dependence of the fluorescence intensity on the composition of water‐alcohol binary solvent systems shows that hydrophobic interactions play an important role in the monomer = multimer equilibrium of ORB in these solutions. The fluorescence lifetimes of ORB in the alcohols are also reported.

Time-correlated photon-counting probe of singlet excitation transport and restricted rotation in Langmuir-Blodgett monolayers

Fluorescence depolarization was monitored by time-correlated single-photon counting in organized monolayers of octadecylrhodamine B (ODRB) in dioleoylphosphatidylcholine (DOL) at air-water interfaces. At low ORDB density, the depolarization was dominated by restricted rotational diffusion. Increases in surface pressure reduced both the angular range and the diffusion constant for rotational motion. At higher ODRB densities, additional depolarization was observed due to electronic excitation transport. A two-dimensional two-particle theory developed by Baumann and Fayer was found to provide an excellent description of the transport dynamics for reduced chromophore densities up to /approximately/ 5.0. The testing of transport theories proves to be relatively insensitive to the orientational distribution assumed for the ODRB transition moments in their two-dimensional systems.