Single injection - 1 mg/kg

Using whole cells in suspension, the cell surface receptors were preincubated for 1h in 96-wells microtiter plates with a high (19xKi) concentration of an antagonist (Spiroxatrine) in order to obtain 95% saturation of antagonist binding to the receptors. The assay volume was 150 μl. Then, the microtiter plates were centrifuged at 3000 rpm (1110g) for 3min.This attached the HEK293 cells with antagonist-blocked receptors to the bottom surface. The microplate was then centrifuged upside-down at 600 rpm (44g) for 10 s to remove the liquid and unbound ligand while retaining cells, including bound ligand, attached to the bottom surface. Immediately, 300 μl of binding buffer (including about 2 nM [3H]-RX821002) was added rather forcefully to the cells. Thereby, most of the HEK293 cells became re-suspended, due to that cell attachment was weak since attachment of the cells had been achieved just by the previous centrifugation in the calcium-free buffer. This step also defines T0 in the on-reaction for the radioligand as well as T0 in the off-reaction for the antagonist. Thereafter, the assays were filtered at different time points. The first filtration was performed after the 30s, then after 2, 6, 15, 36 and 72 min.