Ploy l lysin coated 384 well plate

In total, 5000 HTLA cells were seeded into a white, transparent, and ploy-L-Lysin-coated 384-well plate from PerkinElmer. The cells were co-transfected after 6 h with a plasmid from the PRESTO-Tango GPCR library (Addgene, Watertown, MA, USA, [63 (link)]). We used a mixture of 10 ng plasmid and 0.04 µL Lipofectamine 2000 per well when performing the transfection, as described by [63 (link)]. We used GFP as a transfection control and 100 µM carbachol and the muscarinic M5 receptor as a positive control. After 24 h, the medium was replaced by 45 µL of serum-free medium. The ligand (5 µL) was then added at a final concentration of 30 µM for approximately 24 h. Afterwards, the medium was aspirated, and the cells lysed using 50 µL of bright-Glo reagent (Promega, Madison, WI, USA) diluted 10 times with PBS. After 15 min of incubation with lysis buffer, the luminescence (endpoint, 1500 ms integration time) was measured using a flexstation 3 plate reader.

In total, 5000 HEK293 cells were seeded in a white, transparent and ploy-L-Lysin-coated 384-well plate from PerkinElmer. The cells were co-transfected after 6 h with the cAMP reporter pGL4.29 plasmid (Promega) and a plasmid from the PRESTO-Tango GPCR library (10 ng from each plasmid and 0.04 µL Lipofectamine 2000 per well). As a transfection control, we used GFP and as a positive control we used 10 nM GLP-1 acting on the GLP-1 receptor. After 48 h, the medium was replaced by 45 µL of serum-free medium for one hour. The ligand (5 µL) was then added at a final concentration of 15 µM for 5 h. Afterwards, the medium was aspirated, and the cells were lysed using 50 µL of a mixture of bright-Glo reagent (Promega) diluted 10 times with PBS. After 15 min of incubation with lysis buffer, the luminescence (endpoint, 1500 ms integration time) was determined using a flexstation 3 plate reader.