Real-Time Partitioning of Octadecyl Rhodamine B into Bead-Supported Lipid Bilayer Membranes Revealing Quantitative Differences in Saturable Binding Sites in DOPC and 1:1:1 DOPC/SM/Cholesterol Membranes

Abstract

Quantitative analysis of the staining of cell membranes with the cationic amphimphile, octadecyl rhodamine B (R18) is confounded by probe aggregation and changes to the probes' absorption crosssection and emission quantum yield. In this paper flow cytometry, quantum dot based fluorescence calibration beads and FRET to were used examine real time transfer of octadecyl rhodamine B (R18) from water to two limiting models of the cellular plasma membrane, namely a single component disordered membrane, dioleoyl-L-α-phosphatidylcholine (DOPC), and a ternary mixture of DOPC, cholesterol and sphingomyelin (DSC) membranes, reconstituted on spherical and monodisperse glass beads (lipobeads). The quenching of R18 was analyzed as the probe concentration is raised from 0 to 10 mol% in membranes. The data show a > 2-fold enhancement in the quenching level of the probes that are reconstituted in DSC relative to DOPC membranes at the highest concentration of R18. We have parameterized the propagation of concentration dependent quenching as a function of real-time binding of R18 to lipobeads. In this way, phenomenological kinetics of serum albumin mediated transfer of R18 from the aqueous phase to DOPC and DSC membranes could be evaluated under optimal conditions were the critical aggregation concentration (CAC) of the probe is defined as 14nM. The mass action kinetics of association of R18 with DOPC and DSC lipobeads are shown to be are similar. However, the saturable capacity for accepting exogenous probes is found to be 37% higher in DOPC relative to DSC membranes. The difference is comparable to the disparity in the average molecular areas of DOPC and DSC membranes. Finally this analysis shows little difference in the spectral overlap integrals of the emission spectrum of a fluorescein derivative donor and the absorption spectrum of either monomeric or simulated spectrum of dimeric R18. This approach represents a first step towards a nano-scale probing of membrane heterogeneity in living cells by analyzing differential local FRET among sites of unique receptor expression in living cells.

Recognition of decay accelerating factor and αvβ3 by inactivated hantaviruses: Toward the development of high-throughput screening flow cytometry assays

“…SNV was then transferred to a microfuge tube into which a 0.5 μL aliquot of ≈ 2 mM DMSO-solubilized R18 was added. It is important that the HHB buffer contain 0.1% HSA (97μM), as a large excess of serum albumin is necessary to limit probe aggregation and mediate the transfer of monomeric R18 from the aqueous phase to membranes [23]. The mixture was then vortexed, and incubated at 37°C in the dark under mild vortexing for 30 min.…”

“…The quantum yield of R18 solubilized in lipid bilayer membranes can be correlated to its mole fraction composition in the host membrane [23]. We have developed standard calibration curves to measure and correlate the dequenching of self-quenched R18 to its mole fraction in a single component, dioleoyl-L-α-phosphatidylcholine (DOPC) lipid bilayer membrane, and a 1:1:1 ternary mixture of DOPC/sphingomyelin/cholesterol (DSC) lipid membrane, supported on beads [23].…”

Phase Composition Control in Microsphere-Supported Biomembrane Systems

“…To gain better control of the lipid phase content of PLBs, these microspherical systems enable flow cytometry 37, 38 in concert with fluorescence activated cell sorting (FACS). This widespread technology provides a facile method for purification that is not possible with GUVs, proteoliposomes or other intact biomembrane systems.…”

Comparative Study of Poly (ε-Caprolactone) and Poly(Lactic-co-Glycolic Acid) -Based Nanofiber Scaffolds for pH-Sensing

“…First, a sufficient fluorescence quantum yield allows it to work as a bright internal standard and both its excitation and emission spectrums are far from those of chromoionophore II. Therefore, there is no interference with the chromoionophore II response (44). Also, the signal of R18 will increase slightly in basic conditions.…”