2.5. FLIPR calcium assay

Wildtype HEK293 or C11 cells stably expressing the NMUR2 were seeded at 60 000 cells/well into poly‐L‐lysine (cat no. NC0818114; Sigma Aldrich, Waltham, MA) coated 96 well black clear bottomed cell culture plates (cat no. 07200565, Thermo Fisher Scientific, Waltham, MA) and cultured in complete DMEM with 10% dialyzed FBS. The next day complete media was aspirated and replaced with 50 μL of Hanks balanced salt solution (HBSS) (cat no. 14175095; Gibco, Carlsbad, CA) and cells serum starved for 1 hour in an incubator. 50 μL of 2x FLIPR Calcium 5 dye (cat no. NC9897124, Molecular Devices, Sunnyvale, CA) with 5.0 mmol/L probenecid (final in well concentration of 2.5 mmol/L) was added to the cells and placed back into an incubator for 1 hour. Serial dilutions, 1 nmol/L to 100 μmol/L, of each compound were prepared at 5X final concentration and transferred to a 96 well source plate. Cell and drug plates were placed in a fluorescence imaging plate reader (FLIPR^TETRA) (Molecular Devices, Sunnyvale, CA). The FLIPR^TETRA was programmed to read baseline dye fluorescence for 10s followed by addition of 20 μL (5x) drug/well and read for an additional 120s (acquisition 1 time/s). The maximum (peak) fluorescence observed in each well during the first 40 seconds after compound addition was determined using the FLIPR^TETRA ScreenWorks 4.0 program and results normalized to the average of the baseline fluorescence in each well (first 10 reads). Data from independent experiments (n = 4), conducted in quadruplicate, are presented.