Tristar2 berthold lb942 multimode reader

The determination of GPR55 agonistic activity was carried out using HEK293T-GPR55 cells that overexpress the human GPR55 [62 (link)]. Briefly, HEK293T-GPR55 cells were cultured in 24-well plates (10⁵ cells/well) and transiently transfected with 0.2 µg of the reporter plasmid CRE-Luc that contains six consensus cAMP responsive elements (CRE) linked to firefly luciferase reporter gene using Roti©-Fect (Carl Roth, Karlsruhe, Germany). Transfected cells were treated with increasing concentrations of either the test compounds KIT C and KIT H or the positive controls AM251 and LPI for 6 h. Then, the cells were washed twice with PBS 1× and lysed in 100 µL lysis buffer containing 25 mM Tris-phosphate (pH 7.8), 8 mM MgCl₂, 1 mM DTT, 1% Triton X-100, and 7% glycerol for 15 min at room temperature in a horizontal shaker. Luciferase activity was measured using a TriStar2 Berthold/LB942 multimode reader (Berthold Technologies, Bad Wildbad, Germany) following the instructions of the luciferase assay kit (Promega, Madison, WI, USA). The relative light units (RLUs) were calculated, and the results were expressed as the percentage of activation over the control. The experiment was performed three times.

The determination of GPR55 activity was carried out using the HEK293T-GPR55 cells stably transfected with the human GPR55 cDNA. Briefly, HEK293T-GPR55 cells were transiently transfected with 0.2 µg of the reporter plasmid CRE-Luc that contains six consensus cAMP-responsive elements (CRE) linked to the firefly luciferase reporter gene using Roti©-Fect (Carl Roth, Karlsruhe, Germany). Transfected cells were treated with either VCE-006.1, LPI, or a combination of both. After 6 h of stimulation, cells were washed twice with PBS 1× and lysed in 100 µL lysis buffer containing 25 mM Tris-phosphate (pH 7.8), 8 mM MgCl₂, 1 mM DTT, 1% Triton X-100, and 7% glycerol for 15 min at room temperature in a horizontal shaker. Luciferase activity was measured using a TriStar2 Berthold/LB942 multimode reader (Berthold Technologies, Bad Wildbad, Germany) following the instructions of the luciferase assay kit (Promega, Madison, WI, USA). The RLUs (relative light units) were calculated, and the results were expressed as fold induction over unstimulated cells. The experiment was performed 5–6 times.