Cell Culture Protocols for Kidney and Endothelial Cells

Human kidney cancer cell line HEK293T was obtained from the American Type Culture Collection (ATCC). These cells were grown in DMEM supplemented with 10% v/v fetal bovine serum (FBS). HEK293T cells were seeded into 6-well, 24-well plates or 35mm-dishes coated with fibronectin, collagen and bovine serum albumin (respectively 1 μg/ml, 30 μg/ml and 10 μg/ml in coating medium) [41 (link),42 (link),43 (link)]. Primary human pulmonary arterial endothelial cells (HPAEC) were purchased from Clonetics (San Diego, CA). These were seeded into T-25, T-75 or 6-well plates coated with fibronectin, collagen and bovine serum albumin (respectively 1 μg/ml, 30 μg/ml and 10 μg/ml in coating medium) [41 (link),42 (link),43 (link)]. HPAECs were grown in Medium 200 supplemented with low serum growth supplement LSGS (Cascade Biologics, Carlsbad, CA) and were used between passages 4 and 10 [44 (link)]. The human endothelial cell line EA.hy926 was a gift from Dr. Yang-Ming Yang (Dept. Cell Biology & Anatomy, New York Medical College) and grown in DMEM supplemented with 10% v/v fetal bovine serum and 100 μM hypoxanthine, 0.4 μM aminopterin, and 16 μM thymidine [31 (link)]. Phase contrast microscopy was carried out daily and at the conclusion of each experiment using a Nikon Diaphot Microscope and a Nikon Coolpix digital camera.