Cls3610

HEK293-ΔGαi cells were transfected in suspension with Histamine 3 receptor (H3R) plasmid, pGlosensor-22F (#E2301, Promega) and either a Gα_i/O-NLuc donor plasmid (rescue), wild-type Gα_i-protein plasmid (PC) or Transfection carrier DNA (#E4881; Promega) (NC). Transfected cells were seeded at a density of 3 × 10⁴ cells/well in white 96-well plates with clear flat bottom (#CLS3610, Merck) pre-coated with 100 µg/mL poly-D-lysin (#2780, Merck). Cells were incubated for 48 h at 37 °C and 5% CO2, after which they were washed with CO₂-independent medium (#18045-054, Thermo Fisher Scientific) supplemented with 10% FBS. CO₂-independent medium/10% FBS supplemented with 300 µM 3-isobutyl-1-methylxanthine (#I7018, MERCK) and 2% GloSensor cAMP reagent (#E1291, Promega) was then added to the cells (100 µL/well). After 2 h of incubation at 37 °C, baseline luminescence was measured every 5 s for 30 s using the FLIPR Penta. Thereafter, 25 µL of 5X Histamine (final concentration of 1µM) was automatically added to the cell plate, and bioluminescence was monitored in real time every 5 s for 10 min. Next, 25 µL Forskolin (#F6886, Merck) was added at a final concentration of 5 µM, and bioluminescence was monitored in real time for 40 min every 5 s. Statistical analysis was accomplished with one-way ANOVA followed by Dunnett’s test.

HEK293 cells stably expressing CCR7 were transfected in suspension with pGlosensor-22F. Transfected cells were seeded at a density of 1.5 × 10⁴ cells/well in white, clear flat-bottom 96-well plates (#CLS3610, Merck) coated with 100 µg/mL poly-D-lysin (#2780, Merck, Darmstadt, Germany) and incubated for 48 h at 37 °C and 5% CO₂. Next, cells were washed with CO₂-independent medium (#18045-054, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS and incubated in 100 µL CO₂-independent medium/10%FBS further supplemented with 300 µM 3-isobutyl-1-methylxanthine (#I7018, Merck, Darmstadt, Germany) and 2% GloSensor cAMP reagent (#E1291, Promega, Madison, MD, USA) for 2 h at 37 °C. The plate was transferred to the FLIPR Tetra (Molecular Devices, San Jose, CA, USA) and baseline luminescence was measured for 30 s every 5 s. Thereafter, 25 µL of 5× ligand was added automatically to the cell plate by the FLIPR Tetra and changes in bioluminescence were monitored in real time for 10 min every 5 s. Next, 25 µL Forskolin (#F6886, Merck, Darmstadt, Germany) was added to a final concentration of 5 µM and changes in bioluminescence were monitored in real time for 40 min every 5 s. When required 25 µL, 5× concentrated PTX was added 24 h post-transfection.