Tango Assay for GPR35 Activation

Tango assays were performed in engineered Tango™ U2OS-GPR35-bla cell line (Life Technologies). This cell line allows an endpoint measure of the activity of agonists specific to the GPR35 activation-induced β-arrestin translocation69 (link)70 (link). The cells were passed using McCoy's 5A medium supplemented with 10% dialyzed fetal bovine serum, 0.1 μM NEAA, 25 μM Hepes (pH 7.3), 1 mM sodium pyruvate, 100 U/ml penicillin, 100 μg/ml streptomycin, 200 μg/ml zeocin, 50 μg/ml hygromycin, and 100 μg/ml geneticin in a humidified 37°C/5% CO₂ incubator. For Tango assays, 10000 U2OS-GPR35-bla cells per well were seeded in 384-well, black-wall, clear bottom assay plates with low fluorescence background (Corning). After overnight culture, the cells were stimulated with ligands for 5 hrs at 37°C under 5% CO₂, and then loaded with the cell permeable LiveBLAzer™ FRET (fluorescence resonance energy transfer) B/G substrate. After 2 hr incubation the coumarin to fluorescein ratio was measured using Victor 4 plate reader (PerkinElmer, Waltham, MA, USA). Results obtained were normalized to the maximal response of zaprinast (set to be 100%).