Ebm 2 basal medium

Human umbilical vein endothelial cells (HUVECs) were purchased from Lonza Bioscience. They were cultured in EBM™−2 Basal Medium (Lonza), containing 10% fetal bovine serum and the EGM™−2 SingleQuots™ Supplements (Lonza) required for growth of endothelial cells at 37° C, 5% carbon dioxide. For formation of tubular structure, the 96-well plate was coated with Geltrex™ Matrix (40µL/well) and incubated at 37°C for 30 min. HUVECs (15,000 cells, passage number 9) in 100 µl EGM™−2 were seeded onto the matrix-coated well. Angiogenic vessels formed after 16 hours of incubation were used for drug delivery efficacy examination. Tubulogenesis was confirmed using an inverted microscope (Axio Observer A1, Carl Zeiss, Germany).

LEC proliferation was measured using the BrdU proliferation kit. Briefly, in a flat-bottom 96-well plate 5×10³ LECs were cultured alone in EBM-2 basal medium (Lonza, USA) (negative control), with growth factors [5% FBS, VEGF, FGF, EGF, IGF] (positive control) or with 5×10³ LAD2 cells (stimulated with 10 ng/ml of IL-33 for 3hrs) for 12 hours at 37°C. BrdU label was added after 12 hours and cell cultures were incubated for an extra 12 hours at 37°C. Subsequently, the culture plate was processed according to the manufacturer’s protocol. A SpectraMax Plus 384 Microplate Reader (Molecular Devices, San Jose, CA, USA) was used to measure absorbance at 450/550 nm.

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.