ATP binding cassette (ABC) transmembrane efflux pumps such as P-glycoprotein (ABCB1), multidrug resistance protein 1 (ABCC1), and breast cancer resistance protein (ABCG2) play an important role in anti-cancer drug resistance. A large number of structurally and functionally diverse compounds act as substrates or modulators of these pumps. In vitro assessment of the affinity of drug candidates for multidrug resistance proteins is central to predict in vivo pharmacokinetics and drug–drug interactions. The objective of this study was to identify and characterize new substrates for these transporters. As part of a collaborative project with Life Technologies, 102 fluorescent probes were investigated in a flow cytometric screen of ABC transporters. The primary screen compared substrate efflux activity in parental cell lines with their corresponding highly expressing resistant counterparts. The fluorescent compound library included a range of excitation/emission profiles and required dual laser excitation as well as multiple fluorescence detection channels. A total of 31 substrates with active efflux in one or more pumps and practical fluorescence response ranges were identified and tested for interaction with eight known inhibitors. This screening approach provides an efficient tool for identification and characterization of new fluorescent substrates for ABCB1, ABCC1, and ABCG2.

Hantaviruses cause two severe diseases in humans: hemorrhagic fever with renal syndrome (HFRS) or hantavirus cardio-pulmonary syndrome (HCPS). The lack of vaccines or specific drugs to prevent or treat HFRS and HCPS, and the requirement for conducting experiments in a biosafety level 3 laboratory (BSL-3) limit the ability to probe the mechanism of infection and disease pathogenesis. In this study we have developed a generalizable spectroscopic assay to quantify saturable fluorophore sites solubilized in envelope membranes of Sin Nombre virus (SNV) particles. We then use flow cytometry and live cell confocal fluorescence microscopy imaging to show that UV-killed SNV bind to the cognate receptors of live virions, namely, decay accelerating factor (CD55/DAF) expressed on Tanoue B cells and α v β 3 integrins expressed on Vero E6 cells. SNV binding to DAF is multivalent and of high affinity (K d ≈ 26pM). Selfexchange competition binding assays between fluorescently labeled SNV and unlabeled SNV are used to evaluate an infectious unit-to-particle ratio of ∼1:14000. We have configured the assay for measuring the binding of fluorescently labeled SNV to Tanoue B suspension cells using a high throughput flow cytometer. In this way, we establish a proof of principle high throughput screening assay for binding inhibition. This is a first step towards the development of HTS format assays for small molecule inhibitors of viral-cell interactions, as well as dissecting the mechanism of infection in a BSL-2 environment.

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.