Competitive Binding Assay for CXCR4 Ligands

In order to determine the affinity of the halogenated cyclam derivatives, competitive binding studies were performed with U87-CXCR4 cells using ⁶⁷Ga-Pentixafor (CXCR4 receptor ligand) [26 (link)]. Briefly, triplicate samples containing 0.5 × 10⁶ cells pre-seeded in 6-well plate, about 1.8 kBq of ⁶⁷Ga-Pentixafor (4.0 nM) and 0.001–1000 nM of the tested ligands (total volume 1.0 ml) were incubated at 37 °C for 2 h. After incubation, the cells were washed with 2 × 1 ml of ice-cold tris-buffered saline (pH 7.4) and then collected for measurement with a WIZARD 1480 gamma counter (PerkinElmer, Waltham, MA). The IC₅₀ values were estimated using a least squares fitting routine (GraphPad Prism 6, San Diego, CA, USA).