Hiendoxl endothelial cell growth medium

HEK293T cells (0.1 × 10³) were seeded in a 96-well plate. Cells were transfected with FRG1 expression and silencing vectors, along their respective controls, using Xtremegene9. Transfected cells were grown for 96 h. Afterward, the medium was replaced with fresh medium and 20 µl of MTS reagent (Promega). Absorbance was recorded at 490 nm wavelength using iMark Microplate Reader (Bio–Rad).
HUVECs (0.5 × 10³) were seeded in a 96-well plate in HiEndoXL Endothelial Cell Growth Medium (HiMedia). Post 24 h of seeding, endothelial cell growth medium was replaced with conditioned medium obtained from HEK293T cells transfected with FRG1 expression vector and respective vector control. HUVECs were grown in the respective conditioned medium for 96 h, followed by addition of fresh medium and 20 µl of MTS reagent (Promega).

FRG1 expression vector (pCMV6.XL5.FRG1) and knockdown vector (pLKO.1. FRG1sh) along with their controls were procured from OriGene and Sigma, respectively. Plasmids were purified using Plasmid Mini Kit (Qiagen) using manufacturer’s guidelines. Human embyonic kidney (HEK293T) cells were obtained from National Center for Cell Science (NCCS), Pune, India and were grown in DMEM (PAN-Biotech) with 10% FBS (PAN-Biotech). HEK293T transfection was carried out using X-tremeGENE 9 (Roche) as per manufacturer’s protocol. Human umbilical vein endothelial cells (HUVECs) were procured from HiMedia Laboratories, Mumbai. HUVECs were maintained in HiEndoXL Endothelial Cell Growth Medium (HiMedia).