Efflux assay

Instead of measuring the translocation of a substrate into a compartment, it is sometimes more useful to determine the translocation out of the compartment. Because you can only get something out of a container if something is in it, the compartments are first loaded with 

Once the compartments are loaded, the experimenter removes the substrate that was not taken up (by dialysis, centrifuging, gel filtration, and so on) and uses the loaded compartments in the assay as quickly as possible. The assay typically consists of exposing the loaded compartments to an agent for a certain time and then separating them from the surrounding medium. The amoimt of released substrate is measured (directly or indirectly). Alternatively, the amoimt of substrate remaining in the compartments is measured. 

Hunter, D., and Nathanson, N. (1985). Assay of Muscarinic Acetylcholine Receptor Function in Cultured Cardiac Cells by Stimulation of Rb Efflux, Anal. Biochem. 149 392-398. 

and Cullen, M. (1988). An Isotopic Rubidium Ion Efflux Assay for the Functional Characterization of Nicotinic Acetylcholine Receptors on Clonal Cell Lines, . 4na/, Biochem. 175 212—218. 

and Kaupp, B. (1985). Cyclic GMP Directly Regulates a Cation Conductance in Membranes of Bovine Rods by a Cooperative Mechanism, J. Biol. Chem. 260 6788-6800. 

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In conclusion, there are several drawbacks to the use of Caco-2 cells in studies of active drug transport. Despite these drawbacks, we note that a recent comprehensive study comparing various P-glycoprotein drug efflux assays in drug discovery came to the conclusion that the Caco-2 transport assay is the method of choice, since it displays a biased responsiveness towards compounds with low or moderate permeability - in other words, towards compounds whose intestinal permeability is most likely to be significantly affected by drug efflux mechanisms [101]. 

Chaudhary, KW., O Neal, f.M., Mo, Z.L., Fermini, B., Gallavan, R.H. and Bahinski, A. (2006) Evaluation of the rubidium efflux assay for preclinical identification of hERG blockade. ASSAY and Drug Development Technologies, 4, 73—82. 

The more widely accepted test is the cholesterol efflux assay on cultivated skin fibroblasts. After equilibration with radiolabeled cholesterol, fibroblasts are incubated with albumin in the presence or absence of lipid-free apoA-I. ApoA-I substantially increases cholesterol efflux from normal cells but not from Tangier cells [11, 30]. 

Human skin fibroblasts are cultured from skin biopsy samples. The dermis is cut into small pieces (0.5 mm on each side) and placed into a dish in DMEM containing 10% (v/v) FCS and 1% (v/v) antibiotic-antimycotic solution. When these primary cultures are confluent they are split and cells between passage three and six are used for experiments. For the cholesterol efflux assay, cells are grown in 24-well plates to 60-80% confluence and are labeled with [1,2-3H]-cholesterol (1 pCi/well) for 24 h. Cells are then washed with DMEM and incubated for 4 h at 37°C with DMEM containing BSA (0.2%, v/v) and either 0 (negative control) or 5-30 pg/ml apoA-I. The efflux medium is collected and centrifuged to remove cell debris. Cells are solubilized in 0.1 mol/1 NaOH and the radioactivity in the efflux media and in the cell lysates is determined by scintillation counting [11, 30, 75]. 

Many laboratories use CACO-2 cells as a standard method for assessment of efflux. Since the standard CACO-2 cell assay is very well established, easy to use, reproducible and reliable the corresponding efflux assay can give valuable and helpful data for project support in a screening approach. A prerequisite for the interpretation of efflux data is a characterization of efflux transporters present in the system used and a set of standard efflux markers checked regularly (like digoxin for MDR1). 

As experimental buffer for washing, permeability and efflux assay HBSS, pH 7.4 is proposed. 

An additional experiment is performed usually to determine active efflux. For this experiment flux and efflux assays are performed at 4 °C in comparison to 37 °C. Since efflux is an active process requiring ATP an experiment at 4 °C will show a reduced efflux rate if an active process is involved. 

PURPOSE AND RATIONALE Once a compound is identified in an efflux assays it is sometimes of importance to discriminate between efflux, inhibition potential and to determine the efflux transporter responsible for efflux of the compound. 

Nevertheless, inhibition assays are used very often as a first step before performing more specific efflux assays. 

The greatest precision and best identification is still achieved with cellular efflux assays. However, cellular efflux assays do not have high throughput capacity and... 

The conditions for inhibition assays using CACO-2 cells are as described above for efflux assays. 

In contrast to permeability tests, compounds are added not only to apical compartment (to determine flux A-B) but in an additional experiment to baso-lateral compartment (efflux B-A). As assay time 2h is proposed. At the same time, an inhibitor is added and results of permeability and efflux assays compared to results obtained without inhibitor. Additionally, a selection of inhibitors covering different efflux transporters is used. After a preincubation period used in many studies (15 minutes for example) test compounds are added and flux/efflux studies performed. 

The neurotoxicity of KTX was assayed using rat MCGNs and exhibited potent concentration-dependent toxicity (LD50 value of 3.9 1.9) however, this toxicity showed a unique time delay of 22 h post exposure. Furthermore, KTX toxicity was prevented by co-treatment with NMDA receptor channel antagonists (dextror-phan and MK-801), indicating that KTX-induced neuronal death (morphology and LDH efflux assay) is mediated through an NMDA receptor-dependent mechanism [150]. 

Koh JY, Choi DW (1987) Quantitative determination of glutamate mediated cortical neurontil injury in cell culture by lactate dehydrogenase efflux assay. J Neurosd Methods 20 83—90 Koh JY, Choi DW (1994) Zinc toxicity on cultured cortical neurons involvement of N-methyl-D-aspartate receptors. Neuroscience 60 1049-1057... 

Calcein Efflux Assay 1. Efflux buffer (see Subheading 2.4.1., item 3). 2. [2-(Trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET Anatrace, cat. no. TllOMT) 160 mM in efflux buffer (see Note 3). 3. Sephadex G50. 4. Column for chromatography (1.5 cm i.d.xSO cm length). 

Alternatively, the transporter function can be analyzed by accumulation or efflux assay. Accumulation assay is generally used to determine substrate accumulation rate into the cells mediated by uptake transporters, whereas efflux assay is generally used to determine substrate efflux rate of the cells mediated by efflux transporters. For efflux assay, cells are preincubated with the substrate. 

Gebhard, D., de Morais, S. and Duignan, D.B. (2006) A 96-well efflux assay to identify ABCG2 substrates using a stably transfected MDCK II cell line. Molecular Pharmacology, 3, 45-54. 

The following protocol describes the Rb+ efflux assay for the determination of the hERG blockage [19]. 

Rb+ efflux assay hERG-transfected ICa, (nM) Patch clamp Rb+ efflux assay [20]... 

Note 2 The authors of the reference protocol additionally compared the IC50 values obtained by the rubidium efflux assay, electrophysiology assay, and dofetilide displacement assay for astemizole, E-4031, cisapride, terfenadine, risperidone, quinidine, and sotalol, and the obtained values are demonstrated in Table 1. 

For ion channels, patch-clamping electro-physiological techniques, FRET, and Rh+ efflux assays are used by have limited throughput. The FLIPR has proven useful for nicotinic and P2X receptors (134). 

Rb Drugs (Rb efflux assay) FP (eventually FAAS) 170 25-750 mg L 1 Expert flow system/real-time tsp selection in accordance to the analyte concentration [130]... 

Terstappen, G.C. Functional analysis of native and recombinant ion channels using a high-capacity nonra-dioactive rubidium efflux assay. Anal. Biochem. 1999, 272, 149-155. 

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