PRESTO-Tango GPCR-ome Screening Protocol

The three peptides from human microbiota were synthesized by GenScript Biotech, New Jersey. Screening of compounds in the PRESTO-Tango GPCR-ome was accomplished using previously described methods with several modifications^{57 (link)}. First, HTLA cells were plated in DMEM with 2% dialyzed FBS and 10 U/mL penicillin-streptomycin. Next, the cells were transfected using an in-plate PEI method⁷⁰ . PRESTO-Tango receptor DNAs were resuspended in OptiMEM and hybridized with PEI prior to dilution and distribution into 384-well plates and subsequent addition to cells. After overnight incubation, drugs diluted in DMEM with 1% dialyzed FBS were added to cells without replacement of the medium. After another overnight incubation, the culture medium and peptide-including buffer were removed by aspiration. 20 μl of Bright-Glo solution (Promega) diluted 20-fold in assay buffer was added into each well. The cells were incubated with buffer at room temperature for 15-20 minutes. Then, the luminescence was measured in a Trilux luminescence counter. The relative luminescence units (RLU) were collected from the machine and calculated by fold change based on the basal RLU.