Quest fluo 8 am

Ligand-stimulated intracellular calcium responses were measured in the specified CHO-SecR cell lines, as described previously [30 (link)]. In brief, cells grown to approximately 80% confluence in a 96-well black clear bottom plate were washed with KRH medium containing 2.5 mM probenecid and 0.2% bovine serum albumin, prior to being incubated for 1 h at 37°C in the dark with 0.75 μM of Quest Fluo-8-AM^™ (AAT Bioquest Inc., Sunnyvale, CA). Cells were then washed twice and stimulated by addition of increasing concentrations of potential agonist ligands at 37^oC in a FlexStation 3.0 (Molecular Devices, Sunnyvale, CA). All assays were performed in duplicate and repeated in a minimum of three independent experiments. Responses were quantified based on peak responses in fluorescence emission intensity at 525 nm after exciting the sample at 485 nm for a period of 120 s. Responses were normalized relative to the peak response to natural secretin or to 100 μM ATP, and were plotted using Prism 8 (GraphPad, San Diego, CA).

Secretin was synthesized in our laboratory. Calcitonin and glucagon-like peptide-1 (GLP-1) were purchased from GenScript (Piscataway, NJ). Fmoc amino acids used for peptide synthesis were purchased from Advanced ChemTech (Louisville, KY), and PAL resin was from Sigma-Aldrich (St. Louis, MO). The Gα_s construct was provided by Dr. C. Berlot at Geisinger Clinic in Danville, PA [16 (link)] and the β-arrestin-2-enhanced green fluorescent protein (eGFP) construct was supplied by J. Benovic (Thomas Jefferson University, Philadelphia, PA). Soybean trypsin inhibitor was from Invitrogen (Carlsbad, CA), Fetal Clone II culture medium supplement was from HyClone laboratories (Logan, UT) and bovine serum albumin was from Serologicals Corp. (Norcross, GA). The solid-phase oxidant used in radioiodinations, N-chlorobenzene sulfonamide (Iodobeads), was from Pierce Chemical Co. (Rockford, IL) and the Lance cAMP kit was from PerkinElmer (Waltham, MA). Quest Fluo-8-AM^™ was from AAT Bioquest Inc. (Sunnyvale, CA). All other reagents were of analytical grade.

β-sitosterol and methyl-β-cyclodextrin (MβCD) were from Sigma-Aldrich (St. Louis, MO); Quest Fluo-8-AM^™ was from AAT Bioquest (Sunnyvale, CA); soybean trypsin inhibitor was from Gibco Life Technologies (Grand Island, NY); lipoprotein-deficient serum was from Intracel Resources (Frederick, MD); and bovine serum albumin was from Equitech-Bio (Kerrville, TX). Synthetic CCK-26-33 (CCK-8) was from Bachem (Torrance, CA). All other reagents were analytical grade.