Endothelial growth supplement

HBEC-5i is an immortalized cell line (16 (link)) that has recently been used as an in vitro model for the host-parasite interactions underlying cerebral malaria and sequestration of IE in the human brain (24 (link)– (link)26 (link)). HBEC-5i were seeded onto 50 μg/ml fibronectin (Millipore)-treated flasks and cultured in Dulbecco's modified Eagle medium (DMEM)-F12 medium (Sigma) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM l-glutamine (Invitrogen), 10 μg/ml gentamicin sulfate, and 5 ml endothelial growth supplement (ScienCell) (16 (link)). For adhesion assays, cells were seeded onto fibronectin-coated 8-well chamber slides for a minimum of 48 h prior to the assay (24 (link), 25 (link)). The fibronectin-coated chamber slides were either purchased (BD Biosciences) or coated manually with 50 μg/ml fibronectin (Millipore) for 10 min at 37°C prior to seeding with HBEC-5i. Parasite and HBEC-5i cultures were checked regularly for mycoplasma contamination (27 (link)), and only mycoplasma-negative cultures were used for experiments.

Primary human brain microvascular endothelial cells (HBMEC) and human pulmonary microvascular endothelial cells (HPMEC) were purchased from ScienCell and cultured by following the manufacturer's instructions. Briefly, cells were grown in fibronectin-treated flasks at 37°C in 5% CO₂ using endothelial cell medium (ScienCell) supplemented with 2 mM l-glutamine, 5 ml penicillin-streptomycin (100×), 5 ml endothelial growth supplement (100×; ScienCell), and 5% heat-inactivated fetal bovine serum (Gibco). For adhesion assays, cells were seeded onto fibronectin-coated 8-well chamber slides for a minimum of 72 h prior to the assay, as described above for HBEC-5i. Ring-stage start adhesion assays were carried out as described above, except endothelial cell medium was used rather than DMEM-F12.

Primary human retinal endothelial cells (HREC, Angio-Proteomie) were cultured in endothelial cell media (ECM, ScienCell) containing 5% fetal bovine serum, 1% penicillin/streptromycin (30-002-CI, Corning) with endothelial growth supplement (ScienCell). Human retinal pigment epithelial cells (ARPE-19, ATCC CRL-2302) were cultured in DMEM (Corning) containing 10% fetal bovine serum (FCS500, Excell Bio) and 1% penicillin/streptromycin. Normal medium (low glucose) contained 5 mM D-glucose (G5767, Sigma). High-glucose medium contained 40 mM D-glucose. The HRECs or RPE cells were cultured in normal or high-glucose medium for 6, 24 and 48 hours respectively, and then subjected to experiments.