Top count nxt counter

HEK-293 cells were plated in poly-d-lysine-coated 96-well plates (35.000 cells/well). The following day, cells were transfected in 100 μl transfection medium/well for a total of 5 h and thereafter incubated with 0.5 μCi/ml myo [³H]inositol (Perkin Elmer) in 100 μl growth medium O/N. The subsequent day cells were washed twice with 200 μl/well HBSS (Gibco, Life Technologies) and pre-incubated for 30 min at 37 °C with 100 μl buffer supplemented with 10 mM LiCl. Ligand addition was followed by 120 min incubation at 37 °C. Cells were lysed with 50 μl 10 mM formic acid followed by incubation on ice for 30 min, 20 μl of the extract was transferred to a white 96-well plate and 80 μl of 1:8 diluted YSi SPA scintillation beads (Perkin Elmer) was added. After vigorous shaking, the plate was centrifuged for 5 min at 1500 rpm, and light emission (scintillation) was recorded on a Packard Top Count NXT counter after an 8 h delay. Determinations were made in triplicates.

One day after transfection COS-7 cells were plated into poly-d-lysine-coated 96-well plates (20,000 cells/well) and incubated with 0.5 μCi/ml myo[³H]inositol (Perkin Elmer) in 100 μl growth medium. The following day cells were washed twice with HBSS (Gibco, Life Technologies) and incubated for 30 min 37 °C in 100 μl buffer supplemented with 10 mM LiCl prior to ligand addition followed by 90 min incubation 37 °C. Cells were lysed with 50 μl 10 mM formic acid followed by incubation on ice for 30–60 min 20 μl of the extract were transferred to a white 96-well plate and 80 μl of 1:8 diluted YSi poly-d-lysine coated beads (Perkin Elmer) were added. After vigorously shaking the plate was centrifuged for 5 min at 1500 rpm, and γ-radiation was counted on a Packard Top Count NXT counter after an 8 h delay. Determinations were made in duplicates.

FFAR1 was cloned into the expression vector pCMV-Tag 2B encoding a N-terminal FLAG tag epitope (Stratagene), using the QuikChange method. COS7 cells were transfected using calcium phosphate precipitation method. IP accumulation assays were conducted as described by Hauge et al. (7 (link)). Competition binding assays were performed by adding increasing doses of TAK-875 and Compound 1 to the binding buffer (Hepes wash buffer + 100 µg/mL bacitracin), and immediately after, 50 µL tracer solution containing ³H-TAK-875 (5,000 cpm/well) was added. Plates were incubated for 3 h and washed twice, and γ-radiation was counted on a Packard Top Count NXT counter. Additional details are included in the SI Appendix.