HTLA Cell-Based GPCR Screening Assay

HTLA cells (an HEK293 cell line stably expressing a tTA-dependent luciferase reporter and a β-arrestin2-TEV fusion gene) when coupled with the PRESTO-Tango GPCR-ome screening assay are used as a novel tool to validate GPCR-ligand interactions as previously reported (Kroeze et al., 2015 (link)). HTLA cells were grown in DMEM (11965092, Thermo Fisher Scientific, Grand Island, NY) supplemented with 20% FBS (S11150H, Bio-Techne, Minneapolis, MN), penicillin (100 U ml⁻¹)/streptomycin (0.1 mg ml⁻¹) (15140122, Thermo Fisher Scientific, Grand Island, NY), 2-mM L-glutamine (25030081, Thermo Fisher Scientific, Grand Island, NY) and 100 μg ml⁻¹ Hygromycin B (10687010, Thermo Fisher Scientific, Grand Island, NY) (Dogra et al., 2016 (link); Kroeze et al., 2015 (link)). HTLA cells were plated onto their respective poly-l-lysine (102691, MP Biomedicals, LLC) coated plates prior to treatment. Human umbilical vein endothelial cells (HUVECs) were plated on 0.1% gelatin-coated plates and grown in EBM-2 basal medium (CC-3156, Lonza, Walkersville, MD) supplemented with EGM2 MV SingleQuots (CC-4147, Lonza, Walkersville, MD) using 10% FBS and used between passages 2 and 4.