Poly l lysine (pll)

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.

All ECM protein solutions were added overnight at 4°C to sterile 20 mm round cover glass (Celltreat, 229173) suitable for high-resolution imaging unless otherwise indicated. Fibronectin was purified in-house from bovine plasma^{29 (link)} (Pel-Freez Biologicals 37141–1), sterile filtered with a 0.2 μm filter, and coated at 20 μg/ml in phosphate buffered saline (PBS). Gelatin from bovine skin (Sigma-Aldrich G9391) was dissolved in PBS at 1 mg/ml, autoclaved, and sterile filtered with a 0.2 μm filter. The gelatin solution was added to coverslips without further dilution at room temperature (RT). Coverslips for laminin were precoated with 100 μg/mL poly-L-lysine (MP Biomedicals, 0219454405) for 30 minutes at RT to facilitate binding efficacy per the manufacturer’s recommendation. laminin (Corning, 354232) was added at 20 μg/ml in PBS. Matrigel (Corning, 354230) was diluted to 1:100 (~100 μg/ml) in basal media. Collagen from purified rat tail^{30 (link),31 (link)} was diluted to 50 μg/ml in 30% ethanol and the coverslips were dried overnight in a biological safety cabinet under laminar flow.