Tc lpa5 4

STC-1 cells were seeded into black 96-well plate (5 × 10 4 cells per well) and cell viability was measured as described Santos-Hernández et al. (2018) (link). The cells were exposed to synthetic peptides or GPCR antagonists for 2 h or 1.5h, respectively. After incubation, the medium was aspirated and it was incubated for 1 h with Alamar Blue solution 1:10; v:v, (AlamarBlue™ Cell Viability Reagent, ThermoFisher Scientific, Walthman, USA) prior fluorescence measurement at 590/530 excitation/emission wavelength. Synthetic peptides were tested in HEPES buffer (20 mM HEPES 1 M, 10 mM glucose, 140 mM NaCl, 4.5 mM KCl, 1.2 mM CaCl2, 1.2 mM MgCl2, pH 7.4), all reagents were supplied by Merck KGaA, Darmstadt, Germany. The CaSR antagonist, NPS2143 (Sigma-Aldrich, Sigma Aldrich-Merck KGaA, Darmstadt, Germany), and GPR93 antagonist, TC LPA5 4 (Tocris, Bio-Techne, Minneapolis, USA), were dissolved in dimethylsulfoxide (DMSO, Sigma Aldrich-Merck KGaA, Darmstadt, Germany) at 2% and then diluted with HEPES buffer (1:1, v:v). NPS2143 was tested at 100 µM, 50 µM, 25 µM, 12 µM and 6 µM; and TC LPA5 4 was tested at 8 µM, 4 µM and 2 µM. Cell viability experiments were performed in triplicate and the fluorescence measurement in duplicate.