Radiometric Cellular Assays

Receptor binding or transporter binding assays using radioligands are classical biochemical methods used to characterize and screen these membrane-bound proteins. Although often performed on membrane preparations, these assays can in principle also be performed on whole cells. In its most simple form, the cells are seeded to grow on plates and the medium is exchanged for the appropriate binding buffer when the cells have reached the desired density. The compounds to be screened are added with the radioligand and, after incubation, the bound radioactivity is determined after a number of washing steps and the addition of scintillant. This type of assay is classically performed on ligand-gated transport proteins such as the dopamine transporter [47, 48], but they can also be used for receptor binding or enzymatic studies [49-52]. In the latter case, it should be kept in mind that, due to receptor-internalization, a fraction of the bound radioactivity might not be in equilibrium as it is trapped inside the cell [53]. In addition, a nonspecific trapping of radioactivity within cellular organelles has been described, and this must be considered for certain types of radioligand [54]. As described for the ELISA technology, due to the necessary washing steps these types of assays must be characterized as nonhomogeneous. However, they can be transformed into homogeneous technology when receptor-ligand complexes are detected by scintillation proximity [55, 56]. In contrast to the liquid scintillation system used for filter binding assays, in scintillation proximity only the receptor-bound P-radiation-emitting ligand is able to generate light emission, which can be monitored using a scintillation counter. Depending on the isotope used, the decay generates P-particles, which have a limited travel distance with sufficient energy to excite the scintillant. For whole-cell assays, Scintiplateâ„¢ (PerkinElmer) or Cytostarâ„¢ (GE Helthcare) plates can be used for a pseudohomogeneous spatial separation of the bound/nonbound signals. In such plates, the solid scintillant is incorporated into the plastic; the plates exhibit clear-bottomed wells for microscopic control of cell growth, and are cell-culture-treated for better cell attachment. Cytostar-based assays have been described for bile acid uptake in transfected HEK-293 cells [57], ionotropic glutamate receptors [58, 59], and thymidine incorporation [60]. The similar Scintiplates have been used for radioligand binding studies on whole cells [61].

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